Lipid encapsulating interfering RNA

ABSTRACT

Rich tooling is provided for REST application development that integrates the exploration of a REST API, modeling of data types and the REST API, and the generation of artifacts using the modeled REST API and data types.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.12/852,379, filed Aug. 6, 2010, which is a Divisional of U.S. patentapplication Ser. No. 11/148,152, filed Jun. 7, 2005, which claims thebenefit of U.S. Provisional Patent Application Nos. 60/577,961 filedJun. 7, 2004, 60/578,075 filed Jun. 7, 2004, 60/610,746, filed Sep. 17,2004, and 60/679,427, filed May 9, 2005, the disclosures of each ofwhich are hereby incorporated by reference in their entirety for allpurposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The Sequence Listing written in file086399-001962US-0963783_SequenceListing.txt, created on Nov. 9, 2015,1,226 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for thetherapeutic delivery of a nucleic acid comprising a serum-stable lipiddelivery vehicle encapsulating a nucleic acid to provide efficient RNAinterference (RNAi) in a cell or mammal. More particularly, the presentinvention is directed to using a small interfering RNA (siRNA)encapsulated in a serum-stable lipid particle having a small diametersuitable for systemic delivery.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved, sequencespecific mechanism triggered by double stranded RNA (dsRNA) that inducesdegradation of complementary target single stranded mRNA and “silencing”of the corresponding translated sequences (McManus and Sharp, NatureRev. Genet. 3:737 (2002)). RNAi functions by enzymatic cleavage oflonger dsRNA strands into biologically active “short-interfering RNA”(siRNA) sequences of about 21-23 nucleotides in length (Elbashir, etal., Genes Dev. 15:188 (2001)).

siRNA can be used downregulate or silence the transcription andtranslation of a gene product of interest. For example, it is desirableto downregulate genes associated with liver diseases and disorders suchas hepatits. In particular, it is desirable to downregulate genesassociated with hepatitis viral infection and survival.

An effective and safe nucleic acid delivery system is required forinterference RNA to be therapeutically useful. Viral vectors arerelatively efficient gene delivery systems, but suffer from a variety oflimitations, such as the potential for reversion to the wild type aswell as immune response concerns. As a result, nonviral gene deliverysystems are receiving increasing attention (Worgall, et al., Human GeneTherapy 8:37 (1997); Peeters, et al., Human Gene Therapy 7:1693 (1996);Yei, et al., Gene Therapy 1: 192 (1994); Hope, et al., MolecularMembrane Biology 15:1 (1998)). Furthermore, viral systems are rapidlycleared from the circulation, limiting transfection to “first-pass”organs such as the lungs, liver, and spleen. In addition, these systemsinduce immune responses that compromise delivery with subsequentinjections.

Plasmid DNA-cationic liposome complexes are currently the most commonlyemployed nonviral gene delivery vehicles (Feigner, Scientific American276:102 (1997); Chonn, et al., Current Opinion in Biotechnology 6:698(1995)). For instance, cationic liposome complexes made of anamphipathic compound, a neutral lipid, and a detergent for transfectinginsect cells are disclosed in U.S. Pat. No. 6,458,382. Cationic liposomecomplexes are also disclosed in U.S. Patent Publication No.2003/0073640.

Cationic liposome complexes are large, poorly defined systems that arenot suited for systemic applications and can elicit considerable toxicside effects (Harrison, et al., Biotechniques 19:816 (1995); Li, et al.,The Gene 4:891 (1997); Tam, et al, Gene Ther. 7:1867 (2000)). As large,positively charged aggregates, lipoplexes are rapidly cleared whenadministered in vivo, with highest expression levels observed infirst-pass organs, particularly the lungs (Huang, et al., NatureBiotechnology 15:620 (1997); Templeton, et al., Nature Biotechnology15:647 (1997); Hofland, et al., Pharmaceutical Research 14:742 (1997)).

Other liposomal delivery systems include, for example, the use ofreverse micelles, anionic and polymer liposomes. Reverse micelles aredisclosed in U.S. Pat. No. 6,429,200. Anionic liposomes are disclosed inU.S. Patent Application No. 2003/0026831. Polymer liposomes, thatincorporate dextrin or glycerol-phosphocholine polymers, are disclosedin U.S. Patent Application Nos. 2002/0081736 and 2003/0082103,respectively.

A gene delivery system containing an encapsulated nucleic acid forsystemic delivery should be small (i.e., less than about 100 nmdiameter) and should remain intact in the circulation for an extendedperiod of time in order to achieve delivery to affected tissues. Thisrequires a highly stable, serum-resistant nucleic acid-containingparticle that does not interact with cells and other components of thevascular compartment. The particle should also readily interact withtarget cells at a disease site in order to facilitate intracellulardelivery of a desired nucleic acid.

Recent work has shown that nucleic acids can be encapsulated in small(about 70 nm diameter) “stabilized nucleic acid-lipid particles” (SNALP)that consist of a single plasmid encapsulated within a bilayer lipidvesicle (Wheeler, et al., Gene Therapy 6:271 (1999)). These SNALPstypically contain the “fusogenic” lipid dioleoylphosphatidylethanolamine(DOPE), low levels of cationic lipid, and are stabilized in aqueousmedia by the presence of a poly(ethylene glycol) (PEG) coating. SNALPhave systemic application as they exhibit extended circulation lifetimesfollowing intravenous (i.v.) injection, accumulate preferentially atdistal tumor sites due to the enhanced vascular permeability in suchregions, and can mediate transgene expression at these tumor sites. Thelevels of transgene expression observed at the tumor site following i.v.injection of SPLP containing the luciferase marker gene are superior tothe levels that can be achieved employing plasmid DNA-cationic liposomecomplexes (lipoplexes) or naked DNA.

Thus, there remains a strong need in the art for novel and moreefficient methods and compositions for introducing nucleic acids, suchas interfering RNA, into cells. In addition, there is a need in the artfor methods of treating or preventing disorders such as hepatitis bydownregulating genes associated with viral infection and survival. Thepresent invention addresses this and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention comprises novel, stable nucleic acid-lipidparticles (SNALP) encapsulating one or more interfering RNA molecules,methods of making the SNALPs and methods of deliverubg and/oradministering the SNALPs.

In one embodiment, the invention provides for a nucleic acid-lipidparticle comprising an interfering RNA and a cationic lipid of Formula Ior II and having the following structures:

wherein R¹ and R² are independently selected from the group consistingof: H and C₁-C₃ alkyls; and R³ and R⁴ are independently selected fromthe group consisting of alkyl groups having from about 10 to about 20carbon atoms, wherein at least one of R³ and R⁴ comprises at least twosites of unsaturation. In a preferred embodiment, that cationic lipid isselected from 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA) and1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA). In a preferredembodiment, the interfering RNA molecule is fully encapsulated withinthe lipid bilayer of the nucleic acid-lipid particle such that thenucleic acid in the nucleic acid-lipid particle is resistant in aqueoussolution to degradation by a nuclease. In a preferred embodiment, thenucleic acid particle is substantially non-toxic to mammals. The nucleicacid lipid particles may further comprise a non-cationic lipid, abilayer stabilizing component (i.e., a conjugated lipid that preventsaggregation of particles, a cationic polymer lipid, a sterol (e.g.,cholesterol) and combinations thereof.

In some embodiments, the interfering RNA is a small-interfering RNAmolecule that is less than about 60 nucleotides in length or adouble-stranded RNA greater than about 25 nucleotides in length. In someembodiments the interfering RNA is transcribed from a plasmid, inparticular a plasmid comprising a DNA template of a target sequence.

In one embodiment, the non-cationic lipid is selected fromdistearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), a sterol (e.g.,cholesterol) and a mixture thereof.

In one embodiment, the conjugated lipid that inhibits aggregation ofparticles is one or more of a polyethyleneglycol (PEG)-lipid conjugate,a polyamide (ATTA)-lipid conjugate, and a mixture thereof. In oneaspect, the PEG-lipid conjugate is one or more of a PEG-dialkyloxypropyl(DAA), a PEG-diacylglycerol (DAG), a PEG-phospholipid, a PEG-ceramide,and a mixture thereof. In one aspect, the PEG-DAG conjugate is one ormore of a PEG-dilauroylglycerol (C₁₂), a PEG-dimyristoylglycerol (C₁₄),a PEG-dipalmitoylglycerol (C₁₆), and a PEG-distearoylglycerol (C₁₈). Inone aspect, the PEG-DAA conjugate is one or more of aPEG-dilauryloxypropyl (C₁₂), a PEG-dimyristyloxypropyl (C₁₄), aPEG-dipalmityloxypropyl (C₁₆), and a PEG-distearyloxypropyl (C₁₈).

The nucleic acid-lipid particles of the present invention are useful forthe therapeutic delivery of nucleic acids comprising an interfering RNAsequence. In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease in a mammal bydownregulating or silencing the transcription and translation of atarget nucleic acid sequence of interest. In some embodiments, aninterfering RNA is formulated into a nucleic acid-lipid particle, andthe particles are administered to patients requiring such treatment. Inother embodiments, cells are removed from a patient, the interfering RNAdelivered in vitro, and reinjected into the patient. In one embodiment,the present invention provides for a method of introducing a nucleicacid into a cell by contacting a cell with a nucleic acid-lipid particlecomprised of a cationic lipid, a non-cationic lipid, a conjugated lipidthat inhibits aggregation, and an interfering RNA.

In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the totalinjected dose of the nucleic acid-lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than 20%, 30%, 40% and as much as 60%, 70% or 80% of the totalinjected dose of the nucleic acid-lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In one embodiment, thepresence of an interfering RNA in cells of the lung, liver, tumor or ata site of inflammation is detectable at about 8, 12, 24, 36, 48, 60, 72or 96 hours after administration. In one embodiment, downregulation ofexpression of the target sequence is detectable at about 8, 12, 24, 36,48, 60, 72 or 96 hours after administration. In one embodiment,downregulation of expression of the target sequence occurspreferentially in tumor cells or in cells at a site of inflammation. Inone embodiment, the presence of an interfering RNA in cells at a sitedistal to the site of administration is detectable at least four daysafter intravenous injection of the nucleic acid-lipid particle. Inanother embodiment, the presence of an interfering RNA in of cells inthe lung, liver or a tumor is detectable at least four days afterinjection of the nucleic acid-lipid particle. In another embodiment, thenucleic acid-lipid particle is administered parenterally orintraperitoneally.

The particles are suitable for use in intravenous nucleic acid transferas they are stable in circulation, of a size required forpharmacodynamic behavior resulting in access to extravascular sites andtarget cell populations. The invention also provides forpharmaceutically acceptable compositions comprising a nucleic acid-lipidparticle.

Another embodiment of the present invention provides methods for in vivodelivery of interfering RNA. A nucleic acid-lipid particle comprising acationic lipid, a non-cationic lipid, a conjugated lipid that inhibitsaggregation of particles, and interfering RNA is administered (e.g.,intravenously) to a subject (e.g., a mammal such as a human). In someembodiments, the invention provides methods for in vivo delivery ofinterfering RNA to the liver of a mammalian subject.

A further embodiment of the present invention provides a method oftreating a disease or disorder in a mammalian subject. A therapeuticallyeffective amount of a nucleic acid-lipid particle comprising a cationiclipid, a non-cationic lipid, a conjugated lipid that inhibitsaggregation of particles, and interfering RNA is administered to themammalian subject (e.g., a rodent such as a mouse, a primate such as ahuman or a monkey). In some embodiments, the disease or disorder isassociated with expression and/or overexpression of a gene andexpression or overexpression of the gene is reduced by the interferingRNA.

DEFINITIONS

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids” whichinclude fats and oils as well as waxes; (2) “compound lipids” whichinclude phospholipids and glycolipids; (3) “derived lipids” such assteroids.

“Lipid vesicle” refers to any lipid composition that can be used todeliver a compound including, but not limited to, liposomes, wherein anaqueous volume is encapsulated by an amphipathic lipid bilayer; orwherein the lipids coat an interior comprising a large molecularcomponent, such as a plasmid comprising an interfering RNA sequence,with a reduced aqueous interior; or lipid aggregates or micelles,wherein the encapsulated component is contained within a relativelydisordered lipid mixture.

As used herein, “lipid encapsulated” can refer to a lipid formulationthat provides a compound with full encapsulation, partial encapsulation,or both. In a preferred embodiment, the nucleic acid is fullyencapsulated in the lipid formulation (e.g., to form an SPLP, pSPLP, orother SNALP).

As used herein, the term “SNALP” refers to a stable nucleic acid lipidparticle, including SPLP. A SNALP represents a vesicle of lipids coatinga reduced aqueous interior comprising a nucleic acid (e.g., ssDNA,dsDNA, ssRNA, micro RNA (miRNA), short hairpin RNA (shRNA), dsRNA,siRNA, or a plasmid, including plasmids from which an interfering RNA istranscribed). As used herein, the term “SPLP” refers to a nucleic acidlipid particle comprising a nucleic acid (e.g., a plasmid) encapsulatedwithin a lipid vesicle. SNALPs and SPLPs typically contain a cationiclipid, a non-cationic lipid, and a lipid that prevents aggregation ofthe particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs havesystemic application as they exhibit extended circulation lifetimesfollowing intravenous (i.v.) injection, accumulate at distal sites(e.g., sites physically separated from the administration site and canmediate expression of the transfected gene at these distal sites. SPLPsinclude “pSPLP” which comprise an encapsulated condensing agent-nucleicacid complex as set forth in WO 00/03683.

The term “vesicle-forming lipid” is intended to include any amphipathiclipid having a hydrophobic moiety and a polar head group, and which byitself can form spontaneously into bilayer vesicles in water, asexemplified by most phospholipids.

The term “vesicle-adopting lipid” is intended to include any amphipathiclipid that is stably incorporated into lipid bilayers in combinationwith other amphipathic lipids, with its hydrophobic moiety in contactwith the interior, hydrophobic region of the bilayer membrane, and itspolar head group moiety oriented toward the exterior, polar surface ofthe membrane. Vesicle-adopting lipids include lipids that on their owntend to adopt a nonlamellar phase, yet which are capable of assuming abilayer structure in the presence of a bilayer-stabilizing component. Atypical example is DOPE (dioleoylphosphatidylethanolamine). Bilayerstabilizing components include, but are not limited to, conjugatedlipids that inhibit aggregation of the SNALPs, polyamide oligomers(e.g., ATTA-lipid derivatives), peptides, proteins, detergents,lipid-derivatives, PEG-lipid derivatives such as PEG coupled todialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled tophosphatidyl-ethanolamines, and PEG conjugated to ceramides as describedin U.S. Pat. No. 5,885,613.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Amphipathic lipids are usually the major component of alipid vesicle. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids and sphingolipids. Representative examples of phospholipidsinclude, but are not limited to, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Othercompounds lacking in phosphorus, such as sphingolipid, glycosphingolipidfamilies, diacylglycerols and .beta.-acyloxyacids, are also within thegroup designated as amphipathic lipids. Additionally, the amphipathiclipid described above can be mixed with other lipids includingtriglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.

The term “noncationic lipid” refers to any neutral lipid as describedabove as well as anionic lipids. Non-cationic lipids include, e.g.,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, and1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE).

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerol, cardiolipin, diacylphosphatidylserine,diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “cationic lipid” refers to any of a number of lipid speciesthat carry a net positive charge at a selected pH, such as physiologicalpH. Such lipids include, but are not limited to:1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA) and1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE). The following lipids are cationic and have a positivecharge at below physiological pH: DODAP, DODMA, DMDMA and the like.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a liposome, an SNALP orother drug delivery system to fuse with membranes of a cell. Themembranes can be either the plasma membrane or membranes surroundingorganelles, e.g., endosome, nucleus, etc.

The term “diacylglycerol” refers to a compound having 2-fatty acylchains, R¹ and R², both of which have independently between 2 and 30carbons bonded to the 1- and 2-position of glycerol by ester linkages.The acyl groups can be saturated or have varying degrees ofunsaturation. Diacylglycerols have the following general formula:

The term “dialkyloxypropyl” refers to a compound having 2-alkyl chains,R¹ and R², both of which have independently between 2 and 30 carbons.The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

The term “ATTA” or “polyamide” refers to, but is not limited to,compounds disclosed in U.S. Pat. Nos. 6,320,017 and 6,586,559. Thesecompounds include a compound having the formula

wherein: R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids. Theterm “basic amino acid” refers to naturally-occurring amino acids aswell as synthetic amino acids and/or or amino acid mimetics having a netpositive charge at a selected pH, such as physiological pH. This groupincludes, but is not limited to, lysine, arginine, asparagine,glutamine, histidine and the like. Naturally occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, α-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally occurring amino acid, i.e.,an a carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally occurring amino acid.“Amino acid mimetics” refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

The term “nucleic acid” or “polynucleotide” refers to a polymercontaining at least two deoxyribonucleotides or ribonucleotides ineither single- or double-stranded form. Unless specifically limited, theterms encompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al.(1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. DNA may be in the form ofantisense, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA,product of a polymerase chain reaction (PCR), vectors (P1, PAC, BAC,YAC, artificial chromosomes), expression cassettes, chimeric sequences,chromosomal DNA, or derivatives of these groups. The term nucleic acidis used interchangeably with gene, cDNA, mRNA encoded by a gene, and aninterfering RNA molecule.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor (e.g.,hepatitis virus A, B, C, D, E, or G; or herpes simplex virus).

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence”refers to double-stranded RNA that results in the degradation ofspecific mRNAs and can be used to interfere with translation from adesired mRNA target transcript. Short RNAi that is about 15-30nucleotides in length is referred to as “small-interfering RNA” or“siRNA.” Longer RNAi is generally referred to as “double-stranded RNA”or “dsRNA.” A DNA molecule that transcribes dsRNA or siRNA (forinstance, as a hairpin duplex) also provides RNAi. DNA molecules fortranscribing dsRNA are disclosed in U.S. Pat. No. 6,573,099, and in U.S.Patent Publication Nos. 20020160393 and 20030027783. DNA molecules fortranscribing siRNA are reviewed in Tuschl and Borkhardt, MolecularInterventions, 2:158 (2002).

By “silencing” or “downregulation” of a gene or nucleic acid is intendedto mean a detectable decrease of transcription and/or translation of atarget nucleic acid sequence, i.e., the sequence targeted by the RNAi,or a decrease in the amount or activity of the target sequence orprotein in comparison to the normal level that is detected in theabsence of the interfering RNA or other nucleic acid sequence. Adetectable decrease can be as small as about 5% or 10%, or as great asabout 80%, 90% or 100%. More typically, a detectable decrease is about20%, 30%, 40%, 50%, 60%, or 70%.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles means thatthe particle is not significantly degraded after exposure to a serum ornuclease assay that would significantly degrade free DNA. Suitableassays include, for example, a standard serum assay or a DNAse assaysuch as those described in the Examples below.

“Systemic delivery,” as used herein, refers to delivery that leads to abroad biodistribution of a compound within an organism. Some techniquesof administration can lead to the systemic delivery of certaincompounds, but not others. Systemic delivery means that a useful,preferably therapeutic, amount of a compound is exposed to most parts ofthe body. To obtain broad biodistribution generally requires a bloodlifetime such that the compound is not rapidly degraded or cleared (suchas by first pass organs (liver, lung, etc.) or by rapid, nonspecificcell binding) before reaching a disease site distal to the site ofadministration. Systemic delivery of nucleic acid-lipid particules canbe by any means known in the art including, for example, intravenous,subcutaneous, intraperitoneal, In a preferred embodiment, systemicdelivery of nucleic acid-lipid particles is by intravenous delivery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structures of two exemplary cationic lipids ofthe invention: 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA) and1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

FIG. 2 illustrates the synthetic scheme for DLinDMA.

FIG. 3 illustrates the synthetic scheme for DLenDMA.

FIG. 4 illustrates downregulating β-galactosidase expression inCT26.CL25 cells via in vitro delivery of encapsulatedanti-β-galactosidase siRNA in DSPC:Cholesterol:DODMA:PEG-DMG liposomes.

FIG. 5 illustrates that clearance studies with LUVs showed that SNALPscontaining PEG-DAGs were comparable to SNALPs containingPEG-CeramideC20.

FIG. 6 illustrates the pharmacokinetic properties of SNALPs containingPEG-DAGs.

FIGS. 7A-7D illustrate the biodistribution properties of SNALPscontaining PEG-DAGs.

FIG. 8 illustrates the luciferase gene expression 24 hrs post IVadministration of SPLPs containing PEG-CeramideC₂₀ versus PEG-DAGs inNeuro-2a Tumor Bearing Male A/J Mice.

FIG. 9 illustrates the luciferase gene expression 48 hrs post IVadministration of SPLPs containing PEG-CeramideC₂₀ versus PEG-DAGs inNeuro-2a Tumor Bearing Male A/J Mice.

FIG. 10 illustrates the luciferase gene expression 72 hrs post IVadministration of SPLPs containing PEG-CeramideC₂₀ versus PEG-DAGs inNeuro-2a Tumor Bearing Male A/J Mice.

FIG. 11 illustrates data showing luciferase gene expression in tumors 48hours after intravenous administration of SPLP comprising PEG-DAAconjugates and PEG-DAG conjugates.

FIG. 12 illustrates data showing luciferase gene expression in liver,lung, spleen, heart, and tumor following intravenous administration ofSPLP comprising PEG-DAA conjugates and PEG-DAG conjugates.

FIG. 13 illustrates data from clearance studies in Neuro-2a tumorbearing male A/J mice after administration of SPLPs comprising a PEG-DAAconjugate and containing a plasmid encoding luciferase under the controlof the CMV promoter and SNALPs comprising a PEG-DAA conjugate andcontaining anti-luciferase siRNA.

FIG. 14 illustrates data from studies of the pharmacokinetic propertiesof SPLPs comprising a PEG-DAA conjugate and containing a plasmidencoding luciferase under the control of the CMV promoter and SNALPscomprising a PEG-DAA conjugate and containing anti-luciferase siRNA inNeuro-2a tumor bearing male A/J mice.

FIG. 15 illustrates data from clearance studies in Neuro-2a tumorbearing male A/J mice after administration of SPLPs comprising a PEG-DAAconjugate or a PEG-DAG conjugate and containing a plasmid encodingluciferase under the control of the CMV promoter, pSPLPs comprising aPEG-DAG conjugate and containing a plasmid encoding luciferase under thecontrol of the CMV promoter and SNALPs comprising a PEG-DAA conjugateand containing anti-luciferase siRNA.

FIG. 16 illustrates data from studies of the pharmacokinetic propertiesof SPLPs comprising a PEG-DAA conjugate or a PEG-DAG conjugate andcontaining a plasmid encoding luciferase under the control of the CMVpromoter, pSPLPs comprising a PEG-DAG conjugate and containing a plasmidencoding luciferase under the control of the CMV promoter and SNALPscomprising a PEG-DAA conjugate and containing anti-luciferase siRNA inNeuro-2a tumor bearing male A/J mice.

FIG. 17 illustrates in vitro data demonstrating silencing of luciferaseexpression in luciferase expressing cells treated with SPLPs comprisinga PEG-lipid conjugate and containing a plasmid encoding luciferase underthe control of the CMV promoter and SNALPs comprising a PEG-lipidconjugate and containing anti-luciferase siRNA.

FIG. 18 illustrates in vivo data demonstrating silencing of luciferaseexpression in Neuro-2a tumor bearing male A/J mice treated with SPLPscomprising a PEG-DAA conjugate and containing a plasmid encodingluciferase under the control of the CMV promoter and SNALPs comprising aPEG-DAA conjugate and containing anti-luciferase siRNA.

FIG. 19 illustrates in vivo data demonstrating silencing of luciferaseexpression in Neuro-2a tumor bearing male A/J mice treated with SPLPscomprising a PEG-DAA conjugate and containing a plasmid encodingluciferase under the control of the CMV promoter and SNALPs comprising aPEG-DAA conjugate and containing anti-luciferase siRNA.

FIG. 20 illustrates in vivo data demonstrating silencing of luciferaseexpression in Neuro-2a tumor bearing male A/J mice treated with SPLPscomprising a PEG-DAA conjugate and containing a plasmid encodingluciferase under the control of the CMV promoter and SNALPs comprising aPEG-DAA conjugate and containing anti-luciferase siRNA.

FIG. 21 illustrates in vivo data demonstrating silencing of luciferaseexpression in Neuro-2a tumor bearing male A/J mice treated with SPLPscomprising a PEG-DAA conjugate and containing a plasmid encodingluciferase under the control of the CMV promoter and SNALPs comprising aPEG-DAA conjugate and containing anti-luciferase siRNA.

FIG. 22 illustrates in vivo data demonstrating silencing of luciferaseexpression in Neuro-2a tumor bearing male A/J mice treated with SPLPscomprising a PEG-DAA conjugate and containing a plasmid encodingluciferase under the control of the CMV promoter and SNALPs comprising aPEG-DAA conjugate and containing anti-luciferase siRNA.

FIG. 23 illustrates data showing silencing of gene expression followingin vitro transfection of Neuro2a cells stably expressing luciferase byan SPLP (i.e., SNALP) comprising DODAC, DODMA, or DLinDMA andencapsulating an anti-luciferase siRNA sequence.

FIG. 24 illustrates data showing SNALP-mediated gene silencing in vitro.

FIG. 25 illustrates data showing luciferase gene expression in tumors 48hours following intravenous delivery of SPLP encapsulating a plasmidencoding luciferase. The SPLP comprised PEG-C-DMA conjugates and eitherDODMA or DLinDMA. The PEG moieties had molecular weight of either 2000or 750.

FIG. 26 illustrates data showing luciferase gene expression in Neuro2Atumor bearing male A/J mice 48 hours after intravenous administration ofSPLP encapsulating a plasmid encoding luciferase. The SPLP comprisedvarying percentages (i.e., 15%, 10%, 5% or 2.5%) of PEG-C-DMA and eitherDODMA or DLinDMA.

FIG. 27 illustrates data showing the percentage of the injected dose ofSPLP, SNALP, or empty vesicles remaining in plasma of male A/J micefollowing a single intravenous administration of ³H—CHE-labeled SPLP orSNALP, or empty vesicles, containing various percentages (i.e., 2%, 5%,10%, or 15%) of PEG-C-DMA.

FIG. 28 illustrates data showing the biodistribution SPLP, SNALP orempty vesicles in Neuro-2A tumor-bearing male A/J mice 48 hours after asingle intravenous administration of ³H—CHE-labelled formulationscomprising varying percentages of PEG-C-DMA. The SNALP and emptyvesicles comprised DLinDMA. The SPLP comprised DODMA.

FIG. 29 illustrates data showing silencing of luciferase expression indistal, stable Neuro2A-G tumors in A/J mice 48 hours after intravenousadministration of SNALP comprising DLinDMA.

FIG. 30 illustrates data showing silencing of luciferase expression inNeuro2A-G cells following delivery of SNALP formulations comprisingDLinDMA and encapsulating anti-luciferase siRNA.

FIG. 31 illustrates data showing silencing of luciferase expression inNeuro2A-G cells following delivery of SNALP formulations comprisingDLinDMA and encapsulating anti-luciferase siRNA. Delivery of the SNALPformulations was performed in the absence or presence of chloroquine.

DETAILED DESCRIPTION I. Introduction

The present invention demonstrates the unexpected success ofencapsulating short interfering RNA (siRNA) molecules in SNALPscomprising cationic lipids of Formula I, II, or mixture thereof. TheSNALPs described herein can be used to deliver an siRNA to a cell tosilence a target sequence of interest. SNALP comprising any of a broadrange of concentrations of additional cationic lipids, non-cationiclipids, and other lipids can be used to practice the present invention.The SNALP can be prepared with any nucleic acid comprising aninterfering RNA sequence, from any source and comprising anypolynucleotide sequence, and can be prepared using any of a large numberof methods.

II. Stable Nucleic Acid-Lipid Particles (SNALPs) and Properties Thereof

The stable nucleic acid-lipid particles or, alternatively, SNALPstypically comprise cationic lipid (i.e., a cationic lipid of Formula Ior II) and nucleic acids. Such SNALPs also preferably comprisenoncationic lipid and a bilayer stabilizing component (i.e., aconjugated lipid that inhibits aggregation of the SNALPs). The SNALPs ofthe present invention typically have a mean diameter of about 50 nm toabout 150 nm, more typically about 100 nm to about 130 nm, mosttypically about 110 nm to about 115 nm, and are substantially nontoxic.In addition, the nucleic acids present in the SNALPs of the presentinvention are resistant in aqueous solution to degradation with anuclease.

In one embodiment, the present invention provides stabilized nucleicacid-lipid particles (SPLPs or SNALPs) and other lipid-based carriersystems (e.g., a liposome, a micelle, a virosome, a lipid-nucleic acidparticle, a nucleic acid complex and mixtures thereof) containingcationic lipids of the present invention, i.e., cationic lipids ofFormula I, Formula II, or a combination thereof. The lipid-nucleic acidparticles of the present invention typically comprise a nucleic acid, acationic lipid of Formula I or Formula II, a non-cationic lipid and aPEG-lipid conjugate. The cationic lipid of Formula I or Formula IItypically comprises from about 2% to about 60%, from about 5% to about50%, from about 10% to about 45%, from about 20% to about 40%, or about30% of the total lipid present in said particle. The non-cationic lipidtypically comprises from about 5% to about 90%, from about 10% to about85%, from about 20% to about 80%, from about 30% to about 70%, fromabout 40% to about 60% or about 48% of the total lipid present in saidparticle. The PEG-lipid conjugate typically comprises from about 1% toabout 20%, from about 1.5% to about 18%, from about 4% to about 15%,from about 5% to about 12%, or about 2% of the total lipid present insaid particle. The nucleic acid-lipid particles of the present inventionmay further comprise cholesterol. If present, the cholesterol typicallycomprises from about 10% to about 60%, from about 12% to about 58%, fromabout 20% to about 55%, or about 48% of the total lipid present in saidparticle. It will be readily apparent to one of skill in the art thatthe proportions of the components of the nucleic acid-lipid particlesmay be varied, e.g., using the ERP assay described herein. For examplefor systemic delivery, the cationic lipid may comprise from about 5% toabout 15% of the total lipid present in said particle and for local orregional delivery, the cationic lipid comprises from about 40% to about50% of the total lipid present in said particle.

A. Cationic Lipids

Cationic lipids of Formula I and II may be used in the presentinvention, either alone or in combination with one or more othercationic lipid species or non-cationic lipid species. Cationic lipids ofFormula I and II have the following structures:

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls.R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms; at least one of R³ and R⁴ comprisesat least two sites of unsaturation. In one embodiment, R³ and R⁴ areboth the same, i.e., R³ and R⁴ are both linoleyl (C18), etc. In anotherembodiment, R³ and R⁴ are different, i.e., R³ is myristyl (C14) and R⁴is linoleyl (C18). In a preferred embodiment, the cationic lipids of thepresent invention are symmetrical, i.e., R³ and R⁴ are both the same. Inanother preferred embodiment, both R³ and R⁴ comprise at least two sitesof unsaturation. In some embodiments, R³ and R⁴ are independentlyselected from dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl,and icosadienyl. In a preferred embodiment, R³ and R⁴ are both linoleyl.In some embodiments, R³ and R⁴ comprise at least three sites ofunsaturation and are independently selected from, e.g., dodecatrienyl,tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

The cationic lipids of Formula I and Formula II described hereintypically carry a net positive charge at a selected pH, such asphysiological pH. It has been surprisingly found that cationic lipidscomprising alkyl chains with multiple sites of unsaturation, e.g., atleast two or three sites of unsaturation, are particularly useful forforming lipid-nucleic acid particles with increased membrane fluidity. Anumber of cationic lipids and related analogs, which are also useful inthe present invention, have been described in co-pending U.S. Ser. No.08/316,399; U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185, and WO 96/10390.

Additional suitable cationic lipids include, e.g.,dioctadecyldimethylammonium (“DODMA”), Di stearyldimethylammonium(“DSDMA”), N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”) andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). A number of these lipids and related analogs, whichare also useful in the present invention, have been described in U.S.Pat. Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and5,785,992.

B. Non-Cationic Lipids

The noncationic lipids used in the present invention can be any of avariety of neutral uncharged, zwitterionic or anionic lipids capable ofproducing a stable complex. They are preferably neutral, although theycan alternatively be positively or negatively charged. Examples ofnoncationic lipids useful in the present invention include:phospholipid-related materials, such as lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),di stearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE). Noncationic lipidsor sterols such as cholesterol may be present. Additional nonphosphorouscontaining lipids are, e.g., stearylamine, dodecylamine, hexadecylamine,acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropylmyristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate,alkyl-aryl sulfate polyethyloxylated fatty acid amides,dioctadecyldimethyl ammonium bromide and the like,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, and cerebrosides. Other lipids such aslysophosphatidylcholine and lysophosphatidylethanolamine may be present.Noncationic lipids also include polyethylene glycol-based polymers suchas PEG 2000, PEG 5000 and polyethylene glycol conjugated tophospholipids or to ceramides (referred to as PEG-Cer), as described inco-pending U.S. Ser. No. 08/316,429.

In preferred embodiments, the noncationic lipids arediacylphosphatidylcholine (e.g., distearoylphosphatidylcholine,dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine anddilinoleoylphosphatidylcholine), diacylphosphatidylethanolamine (e.g.,dioleoylphosphatidylethanolamine andpalmitoyloleoylphosphatidylethanolamine), ceramide or sphingomyelin. Theacyl groups in these lipids are preferably acyl groups derived fromfatty acids having C₁₀-C₂₄ carbon chains. More preferably the acylgroups are lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. Inparticularly preferred embodiments, the noncationic lipid will becholesterol, 1,2-sn-dioleoylphosphatidylethanolamine, or eggsphingomyelin (ESM).

C. Bilayer Stabilizing Component

In addition to cationic and non-cationic lipids, the SPLPs of thepresent invention comprise bilayer stabilizing component (BSC) such asan ATTA-lipid or a PEG-lipid, such as PEG coupled to dialkyloxypropyls(PEG-DAA) as described in, e.g., WO 05/026372, PEG coupled todiacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent PublicationNos. 20030077829 and 2005008689), PEG coupled tophosphatidylethanolamine (PE) (PEG-PE), or PEG conjugated to ceramides,or a mixture thereof (see, U.S. Pat. No. 5,885,613). In one preferredembodiment, the BSC is a conjugated lipid that inhibits aggregation ofthe SPLPs. Suitable conjugated lipids include, but are not limited toPEG-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipidconjugates (CPLs) or mixtures thereof. In one preferred embodiment, theSPLPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugatetogether with a CPL.

PEG is a polyethylene glycol, a linear, water-soluble polymer ofethylene PEG repeating units with two terminal hydroxyl groups. PEGs areclassified by their molecular weights; for example, PEG 2000 has anaverage molecular weight of about 2,000 daltons, and PEG 5000 has anaverage molecular weight of about 5,000 daltons. PEGs are commerciallyavailable from Sigma Chemical Co. and other companies and include, forexample, the following: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S—NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM). Inaddition, monomethoxypolyethyleneglycol-acetic acid (MePEG-CH₂COOH), isparticularly useful for preparing the PEG-lipid conjugates including,e.g., PEG-DAA conjugates.

In a preferred embodiment, the PEG has an average molecular weight offrom about 550 daltons to about 10,000 daltons, more preferably of about750 daltons to about 5,000 daltons, more preferably of about 1,000daltons to about 5,000 daltons, more preferably of about 1,500 daltonsto about 3,000 daltons and, even more preferably, of about 2,000daltons, or about 750 daltons. The PEG can be optionally substituted byan alkyl, alkoxy, acyl or aryl group. PEG can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, etc. as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated topolyethyleneglycol to form the bilayer stabilizing component. Suchphosphatidylethanolamines are commercially available, or can be isolatedor synthesized using conventional techniques known to those of skilledin the art. Phosphatidylethanolamines containing saturated orunsaturated fatty acids with carbon chain lengths in the range of C₁₀ toC₂₀ are preferred. Phosphatidylethanolamines with mono- or diunsaturatedfatty acids and mixtures of saturated and unsaturated fatty acids canalso be used. Suitable phosphatidylethanolamines include, but are notlimited to, the following: dimyristoylphosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE) anddistearoylphosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” refers to, but is not limited to,compounds disclosed in U.S. Pat. Nos. 6,320,017 and 6,586,559. Thesecompounds include a compound having the formula

wherein: R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” refers to a compound having 2-fatty acylchains, le and R², both of which have independently between 2 and 30carbons bonded to the 1- and 2-position of glycerol by ester linkages.The acyl groups can be saturated or have varying degrees ofunsaturation. Diacylglycerols have the following general formula:

The term “dialkyloxypropyl” refers to a compound having 2-alkyl chains,R¹ and R², both of which have independently between 2 and 30 carbons.The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In one preferred embodiment, the PEG-lipid is a PEG-DAA conjugate hasthe following formula:

In Formula VI, R¹ and R² are independently selected and are long-chainalkyl groups having from about 10 to about 22 carbon atoms. Thelong-chain alkyl groups can be saturated or unsaturated. Suitable alkylgroups include, but are not limited to, lauryl (C12), myristyl (C14),palmityl (C16), stearyl (C18) and icosyl (C20). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula VI above, “R¹ and R²” are independently selected and arealkyl groups having from about 10 to about 20 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester-containing linker moiety asdescribed above. Suitable alkyl groups include, but are not limited to,lauryl (C12), myristyl (C14), palmityl (C16), stearyl (C18) and icosyl(C20). In a preferred embodiment; R¹ and R² are the same, i.e., they areboth myristyl (C14) or both palmityl (C16) or both stearyl (C18). In apreferred embodiment, the alkyl groups are saturated.

In Formula VI above, “PEG” is a polyethylene glycol having an averagemolecular weight ranging of about 550 daltons to about 10,000 daltons,more preferably of about 750 daltons to about 5,000 daltons, morepreferably of about 1,000 daltons to about 5,000 daltons, morepreferably of about 1,500 daltons to about 3,000 daltons and, even morepreferably, of about 2,000 daltons, or about 750 daltons. The PEG can beoptionally substituted with alkyl, alkoxy, acyl or aryl. In a preferredembodiment, the terminal hydroxyl group is substituted with a methoxy ormethyl group.

In Formula VI, above, “L” is a non-ester containing linker moiety or anester containing linker moiety. In a preferred embodiment, L is anon-ester containing linker moiety. Suitable non-ester containinglinkers include, but are not limited to, an amido linker moiety, anamino linker moiety, a carbonyl linker moiety, a carbamate linkermoiety, a urea linker moiety, an ether linker moiety, a disulphidelinker moiety, a succinamidyl linker moiety and combinations thereof. Ina preferred embodiment, the non-ester containing linker moiety is acarbamate linker moiety (i.e., a PEG-C-DAA conjugate). In anotherpreferred embodiment, the non-ester containing linker moiety is an amidolinker moiety (i.e., a PEG-A-DAA conjugate). In a preferred embodiment,the non-ester containing linker moiety is a succinamidyl linker moiety(i.e., a PEG-S-DAA conjugate).

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate and urea linkages. T hose of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992), Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

In a presently preferred embodiment, the PEG-DAA conjugate is adilauryloxypropyl (C12)-PEG conjugate, dimyristyloxypropyl (C14)-PEGconjugate, a dipalmitoyloxypropyl (C16)-PEG conjugate or adisteryloxypropyl (C18)-PEG conjugate. Those of skill in the art willreadily appreciate that other dialkyloxypropyls can be used in thePEG-DAA conjugates of the present invention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses, such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the SNALPs and SPLPs of thepresent invention can further comprise cationic poly(ethylene glycol)(PEG) lipids, or CPLs, that have been designed for insertion into lipidbilayers to impart a positive charge (see, Chen, et al., Bioconj. Chem.11:433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use in the presentinvention, and methods of making and using SPLPs and SPLP-CPLs, aredisclosed, e.g., in U.S. Pat. No. 6,852,334 and WO 00/62813. Cationicpolymer lipids (CPLs) useful in the present invention have the followingarchitectural features: (1) a lipid anchor, such as a hydrophobic lipid,for incorporating the CPLs into the lipid bilayer; (2) a hydrophilicspacer, such as a polyethylene glycol, for linking the lipid anchor to acationic head group; and (3) a polycationic moiety, such as a naturallyoccurring amino acid, to produce a protonizable cationic head group.

Suitable CPL include compounds of Formula VII:A-W—Y  (VII)wherein A, W and Y are as described below.

With reference to Formula VII, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid or a hydrophobic lipid that acts as alipid anchor. Suitable lipid examples include vesicle-forming lipids orvesicle adopting lipids and include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N—N-dialkylaminos,1,2-diacyloxy-3-aminopropanes and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer, such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof about 250 to about 7000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of liposomeapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire liposome moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theliposome moiety e.g., a charge spike. If the charge density isdistributed on the liposome, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A,” and the nonimmunogenic polymer “W,” can be attached byvarious methods and preferably, by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, U.S. Pat.Nos. 6,320,017 and 6,586,559), an amide bond will form between the twogroups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

D. Nucleic Acid Component

The nucleic acid component of the present invention comprises aninterfering RNA that silences (e.g., partially or completely inhibits)expression of a gene of interest. An interfering RNA can be provided inseveral forms. For example an interfering RNA can be provided as one ormore isolated small-interfering RNA (siRNA) duplexes, longerdouble-stranded RNA (dsRNA) or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. The interfering RNA can beadministered alone or in combination with the administration ofconventional agents used to treat the disease or disorder associatedwith the gene of interest. Genes of interest include, but are notlimited to, genes associated with viral infection and survival, genesassociated with liver and kidney diseases and disorders, genesassociated with tumorigenesis and cell transformation, angiogenic genes,immunomodulator genes, such as those associated with inflammatory andautoimmune responses, ligand receptor genes, and genes associated withneurodegenerative disorders.

1. Selecting siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir, et al., Nature411:494-498 (2001) and Elbashir, et al., EMBO J 20: 6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech. 22(3):326-330 (2004).

Typically, the sequence within about 50 to about 100 nucleotides 3′ ofthe AUG start codon of a transcript from the target gene of interest isscanned for dinucleotide sequences (e.g., AA, CC, GG, or UU) (see, e.g.,Elbashir, et al., EMBO J 20: 6877-6888 (2001)). The nucleotidesimmediately 3′ to the dinucleotide sequences are identified as potentialsiRNA target sequences. Typically, the 19, 21, 23, 25, 27, 29, 31, 33,35 or more nucleotides immediately 3′ to the dinucleotide sequences areidentified as potential siRNA target sites. In some embodiments, thedinucleotide sequence is an AA sequence and the 19 nucleotidesimmediately 3′ to the AA dinucleotide are identified as a potentialsiRNA target site. Typically siRNA target sites are spaced at differentpositions along the length of the target gene. To further enhancesilencing efficiency of the siRNA sequences, potential siRNA targetsites may be further analyzed to identify sites that do not containregions of homology to other coding sequences. For example, a suitablesiRNA target site of about 21 base pairs typically will not have morethan 16-17 contiguous base pairs of homology to other coding sequences.If the siRNA sequences are to be expressed from an RNA Pol III promoter,siRNA target sequences lacking more than 4 contiguous A's or T's areselected.

Once the potential siRNA target site has been identified siRNA sequencescomplementary to the siRNA target sites may be designed. To enhancetheir silencing efficiency, the siRNA sequences may also be analyzed bya rational design algorithm to identify sequences that have one or moreof the following features: (1) G/C content of about 25% to about 60%G/C; (2) at least 3 A/Us at positions 15-19 of the sense strand; (3) nointernal repeats; (4) an A at position 19 of the sense strand; (5) an Aat position 3 of the sense strand; (6) a U at position 10 of the sensestrand; (7) no G/C at position 19 of the sense strand; and (8) no G atposition 13 of the sense strand. siRNA design tools that incorporatealgorithms that assign suitable values of each of these features and areuseful for selection of siRNA can be found at, e.g.,http://boz094.ust.hk/RNAi/siRNA.

In some embodiments, once a potential siRNA sequence has beenidentified, the sequence is analyzed for the presence or absence ofimmunostimulatory motifs (e.g., GU-rich motifs) as described in, e.g.,co-pending U.S. Provisional Patent Application No. 60/585,301, filedJul. 2, 2004; 60/589,363, filed Jul. 19, 2004; 60/627,326, filed Nov.12, 2004; and 60/665,297, filed Mar. 25, 2005. Once identified, theimmunostimulatory siRNA molecules can be modified to increase ordecrease their immunostimulatory properties and thenon-immunostimulatory molecules can be modified so that they possessimmunostimulatory properties

2. Generating siRNA

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, longer double-strandedRNA (dsRNA) or as siRNA or dsRNA transcribed from a transcriptionalcassette in a DNA plasmid. siRNA may also be chemically synthesized.Preferably, the synthesized or transcribed siRNA have 3′ overhangs ofabout 1-4 nucleotides, preferably of about 2-3 nucleotides and 5′phosphate termini. The siRNA sequences may have overhangs (e.g., 3′ or5′ overhangs as described in (Elbashir, et al., Genes Dev. 15:188(2001); Nykänen, et al., Cell 107:309 (2001)) or may lack overhangs(i.e., to have blunt ends).

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA); or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Alternatively, one or more DNA plasmids encoding one or more siRNAtemplates are used to provide siRNA. siRNA can be transcribed assequences that automatically fold into duplexes with hairpin loops fromDNA templates in plasmids having RNA polymerase III transcriptionalunits, for example, based on the naturally occurring transcription unitsfor small nuclear RNA U6 or human RNase P RNA H1 (see, Brummelkamp, etal., Science 296:550 (2002); Donzé, et al., Nucleic Acids Res. 30:e46(2002); Paddison, et al., Genes Dev. 16:948 (2002); Yu, et al., Proc.Natl. Acad. Sci. 99:6047 (2002); Lee, et al., Nat. Biotech. 20:500(2002); Miyagishi, et al., Nat. Biotech. 20:497 (2002); Paul, et al.,Nat. Biotech. 20:505 (2002); and Sui, et al., Proc. Natl. Acad. Sci.99:5515 (2002)). Typically, a transcriptional unit or cassette willcontain an RNA transcript promoter sequence, such as an H1-RNA or a U6promoter, operably linked to a template for transcription of a desiredsiRNA sequence and a termination sequence, comprised of 2-3 uridineresidues and a polythymidine (T5) sequence (polyadenylation signal)(Brummelkamp, Science, supra). The selected promoter can provide forconstitutive or inducible transcription. Compositions and methods forDNA-directed transcription of RNA interference molecules is described indetail in U.S. Pat. No. 6,573,099. The transcriptional unit isincorporated into a plasmid or DNA vector from which the interfering RNAis transcribed. Plasmids suitable for in vivo delivery of geneticmaterial for therapeutic purposes are described in detail in U.S. Pat.Nos. 5,962,428 and 5,910,488. The selected plasmid can provide fortransient or stable delivery of a target cell. It will be apparent tothose of skill in the art that plasmids originally designed to expressdesired gene sequences can be modified to contain a transcriptional unitcassette for transcription of siRNA.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (seeU.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994)).

A suitable plasmid is engineered to contain, in expressible form, atemplate sequence that encodes a partial length sequence or an entirelength sequence of a gene product of interest. Template sequences canalso be used for providing isolated or synthesized siRNA and dsRNA.Generally, it is desired to downregulate or silence the transcriptionand translation of a gene product of interest.

3. Genes of Interest

Genes of interest include, but are not limited to, genes associated withviral infection and survival, genes associated with metabolic diseasesand disorders (e.g., liver diseases and disorders), genes associatedwith tumorigenesis and cell transformation, angiogenic genes,immunomodulator genes, such as those associated with inflammatory andautoimmune responses, ligand receptor genes, and genes associated withneurodegenerative disorders.

a) Genes Associated with Viral Infection and Survival

Genes associated with viral infection and survival include thoseexpressed by a virus in order to bind, enter and replicate in a cell. Ofparticular interest are viral sequences associated with chronic viraldiseases. Viral sequences of particular interest include sequences ofHepatitis viruses (Hamasaki, et al., FEBS Lett. 543:51 (2003); Yokota,et al., EMBO Rep. 4:602 (2003); Schlomai, et al., Hepatology 37:764(2003); Wilson, et al., Proc. Natl. Acad. Sci. 100:2783 (2003); Kapadia,et al., Proc. Natl. Acad. Sci. 100:2014 (2003); and FIELDS VIROLOGY(Knipe et al. eds. 2001)), Human Immunodeficiency Virus (HIV) (Banerjea,et al., Mol. Ther. 8:62 (2003); Song, et al., J. Virol. 77:7174 (2003);Stephenson JAMA 289:1494 (2003); Qin, et al., Proc. Natl. Acad. Sci.100:183 (2003)), Herpes viruses (Jia, et al., J. Virol. 77:3301 (2003)),and Human Papilloma Viruses (HPV) (Hall, et al., J. Virol. 77:6066(2003); Jiang, et al., Oncogene 21:6041 (2002)). Exemplary hepatitisviral nucleic acid sequences that can be silenced include, but are notlimited to: nucleic acid sequences involved in transcription andtranslation (e.g., En1, En2, X, P), nucleic acid sequences encodingstructural proteins (e.g., core proteins including C and C-relatedproteins; capsid and envelope proteins including S, M, and/or Lproteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY, 2001,supra). Exemplary Hepatits C nucleic acid sequences that can be silencedinclude, but are not limited to: serine proteases (e.g., NS3/NS4),helicases (e.g. NS3), polymerases (e.g., NS5B), and envelope proteins(e.g., E1, E2, and p7). Hepatitis A nucleic acid sequences are set forthin e.g., Genbank Accession No. NC_001489; Hepatitis B nucleic acidsequences are set forth in, e.g., Genbank Accession No. NC_003977;Hepatitis C nucleic acid sequences are set forth in, e.g., GenbankAccession No. NC_004102; Hepatitis D nucleic acid sequence are set forthin, e.g., Genbank Accession No. NC_001653; Hepatitis E nucleic acidsequences are set forth in e.g., Genbank Accession No. NC_001434; andHepatitis G nucleic acid sequences are set forth in e.g., GenbankAccession No. NC_001710.

b) Genes Associated with Metabolic Diseases and Disorders

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, for example genes expressed in, for example, dyslipidemia(e.g., liver X receptors (e.g., LXRα and LXRβ Genback Accession No.NM_007121), farnesoid X receptors (FXR) (Genbank Accession No.NM_005123), sterol-regulatory element binding protein (SREBP), Site-1protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGcoenzyme-A reductase), Apolipoprotein (ApoB), and Apolipoprotein (ApoE))and diabetes (e.g., Glucose 6-phosphatase) (see, e.g., Forman et al.,Cell 81:687 (1995); Seol et al., Mol. Endocrinol. 9:72 (1995), Zavackiet al., PNAS USA 94:7909 (1997); Sakai, et al., Cell 85:1037-1046(1996); Duncan, et al., J. Biol. Chem. 272:12778-12785 (1997); Willy, etal., Genes Dev. 9(9):1033-45 (1995); Lehmann, et al., J. Biol. Chem.272(6):3137-3140 (1997); Janowski, et al., Nature 383:728-731 (1996);Peet, et al., Cell 93:693-704 (1998)). One of skill in the art willappreciate that genes associated with metabolic diseases and disorders(e.g., diseases and disorders in which the liver is a target and liverdiseases and disorders) include genes that are expressed in the liveritself as well as and genes expressed in other organs and tissues.

c) Genes Associated with Tumorigenesis

Examples of gene sequences associated with tumorigenesis and celltransformation include translocation sequences such as MLL fusion genes,BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002); Scherr, et al., Blood101:1566), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO andAML1-MTG8 (Heidenreich, et al., Blood 101:3157 (2003)); overexpressedsequences such as multidrug resistance genes (Nieth, et al., FEBS Lett.545:144 (2003); Wu, et al, Cancer Res. 63:1515 (2003)), cyclins (Li, etal., Cancer Res. 63:3593 (2003); Zou, et al., Genes Dev. 16:2923(2002)), beta-Catenin (Verma, et al., Clin Cancer Res. 9:1291 (2003)),telomerase genes (Kosciolek, et al., Mol Cancer Ther. 2:209 (2003)),c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al. Exp. Cell Res. 285:39(2003)); and mutated sequences such as RAS (reviewed in Tuschl andBorkhardt, Mol. Interventions, 2:158 (2002)). For example, silencing ofsequences that encode DNA repair enzymes find use in combination withthe administration of chemotherapeutic agents (Collis, et al., CancerRes. 63:1550 (2003)). Genes encoding proteins associated with tumormigration are also target sequences of interest, for example, integrins,selectins and metalloproteinases. The foregoing examples are notexclusive. Any whole or partial gene sequence that facilitates orpromotes tumorigenesis or cell transformation, tumor growth or tumormigration can be included as a gene sequence of interest.

d) Angiogenic/Anti-Angiogenic Genes

Angiogenic genes are able to promote the formation of new vessels. Ofparticular interest is Vascular Endothelial Growth Factor (VEGF) (Reich,et al., Mol. Vis. 9:210 (2003)) or VEGFr. siRNA sequences that targetVEGFr are set forth in, e.g., GB 2396864; U.S. Patent Publication No.20040142895; and CA2456444.

Anti-angiogenic genes are able to inhibit neovascularization. Thesegenes are particularly useful for treating those cancers in whichangiogenesis plays a role in the pathological development of thedisease. Examples of anti-angiogenic genes include, but are not limitedto, endostatin (see e.g., U.S. Pat. No. 6,174,861), angiostatin (see,e.g., U.S. Pat. No. 5,639,725), and VEGF-R2 (see e.g., Decaussin et al.(1999) J. Pathol. 188(4): 369-737).

e) Immonomodulator Genes

Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include cytokines such asgrowth factors (e.g., TGF-α, TGF-□β, EGF, FGF, IGF, NGF, PDGF, CGF,GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-3, IL-4, IL-6, IL-7,IL-10, IL-12, IL-15, IL-20, etc.), interferons (e.g., IFN-α, IFN-β,IFN-γ, etc.), TNF (e.g., TNF-α), and Flt3-Ligand. Fas and Fas Ligandgenes are also immunomodulator target sequences of interest (Song, etal., Nat. Med. 9:347 (2003)). Genes encoding secondary signalingmolecules in hematopoietic and lymphoid cells are also included in thepresent invention, for example, Tec family kinases, such as Bruton'styrosine kinase (Btk) (Heinonen, et al., FEBS Lett. 527:274 (2002)).

f) Cell Receptor Ligands

Cell receptor ligands include ligands that are able to bind to cellsurface receptors (e.g., insulin receptor, EPO receptor, G-proteincoupled receptors, receptors with tyrosine kinase activity, cytokinereceptors, growth factor receptors, etc.), to modulate (e.g., inhibit,activate, etc.) the physiological pathway that the receptor is involvedin (e.g., glucose level modulation, blood cell development, mitogenesis,etc.). Examples of cell receptor ligands include cytokines, growthfactors, interleukins, interferons, erythropoietin (EPO), insulin,glucagon, G-protein coupled receptor ligands, etc.). Templates codingfor an expansion of trinucleotide repeats (e.g., CAG repeats), find usein silencing pathogenic sequences in neurodegenerative disorders causedby the expansion of trinucleotide repeats, such as spinobulbularmuscular atrophy and Huntington's Disease (Caplen, et al., Hum. Mol.Genet. 11:175 (2002)).

g) Tumor Suppressor Genes

Tumor suppressor genes are genes that are able to inhibit the growth ofa cell, particularly tumor cells. Thus, delivery of these genes to tumorcells is useful in the treatment of cancers. Tumor suppressor genesinclude, but are not limited to, p53 (Lamb et al., Mol. Cell. Biol.6:1379-1385 (1986), Ewen et al., Science 255:85-87 (1992), Ewen et al.(1991) Cell 66:1155-1164, and Hu et al., EMBO J. 9:1147-1155 (1990)),RB1 (Toguchida et al. (1993) Genomics 17:535-543), WT1 (Hastie, N. D.,Curr. Opin. Genet. Dev. 3:408-413 (1993)), NF1 (Trofatter et al., Cell72:791-800 (1993), Cawthon et al., Cell 62:193-201 (1990)), VHL (Latifet al., Science 260:1317-1320 (1993)), APC (Gorden et al., Cell66:589-600 (1991)), DAP kinase (see e.g., Diess et al. (1995) Genes Dev.9: 15-30), p16 (see e.g., Marx (1994) Science 264(5167): 1846), ARF (seee.g., Quelle et al. (1995) Cell 83(6): 993-1000), Neurofibromin (seee.g., Huynh et al. (1992) Neurosci. Lett. 143(1-2): 233-236), and PTEN(see e.g., Li et al. (1997) Science 275(5308): 1943-1947).

III. Preparation of SNALPS

The present invention provides a method of preparing serum-stablenucleic acid-lipid particles in which the plasmid or other nucleic acidis encapsulated in a lipid bilayer and is protected from degradation.The particles made by the methods of this invention typically have asize of about 50 nm to about 150 nm, more typically about 100 nm toabout 130 nm, most typically about 110 nm to about 115 nm. The particlescan be formed by any method known in the art including, but not limitedto: a continuous mixing method, a detergent dialysis method, or amodification of a reverse-phase method which utilizes organic solventsto provide a single phase during mixing of the components.

In preferred embodiments, the cationic lipids are lipids of Formula Iand II or combinations thereof. In other preferred embodiments, thenoncationic lipids are ESM, DOPE, DOPC, DPPE, DMPE, 16:0 MonomethylPhosphatidylethanolamine, 16:0 Dimethyl Phosphatidylethanolamine, 18:1Trans Phosphatidylethanolamine, 18:0 18:1 Phosphatidylethanolamine(SOPE), 16:0 18:1 Phosphatidylethanolamine, DSPE, polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls),distearoylphosphatidylcholine (DSPC), cholesterol, or combinationsthereof. In still other preferred embodiments, the organic solvents aremethanol, chloroform, methylene chloride, ethanol, diethyl ether orcombinations thereof.

In a particularly preferred embodiment, the nucleic acid is a plasmid;the cationic lipid is a lipid of Formula I or II or combinationsthereof; the noncationic lipid is ESM, DOPE, PEG-DAAs,distearoylphosphatidylcholine (DSPC), cholesterol, or combinationsthereof (e.g. DSPC and PEG-DAAs); and the organic solvent is methanol,chloroform, methylene chloride, ethanol, diethyl ether or combinationsthereof

In a particularly preferred embodiment, the present invention providesfor nucleic acid-lipid particles produced via a continuous mixingmethod, e.g., process that includes providing an aqueous solutioncomprising a nucleic acid such as an siRNA or a plasmid, in a firstreservoir, and providing an organic lipid solution in a secondreservoir, and mixing the aqueous solution with the organic lipidsolution such that the organic lipid solution mixes with the aqueoussolution so as to substantially instantaneously produce a liposomeencapsulating the nucleic acid (e.g., siRNA). This process and theapparatus for carrying this process is described in detail in U.S.Patent Publication No. 20040142025.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a liposome substantially instantaneously upon mixing. As usedherein, the phrase “continuously diluting a lipid solution with a buffersolution” (and variations) generally means that the lipid solution isdiluted sufficiently rapidly in a hydration process with sufficientforce to effectuate vesicle generation. By mixing the aqueous solutioncomprising a nucleic acid with the organic lipid solution, the organiclipid solution undergoes a continuous stepwise dilution in the presenceof the buffer solution (i.e., aqueous solution) to produce a nucleicacid-lipid particle.

The serum-stable nucleic acid-lipid particles formed using thecontinuous mixing method typically have a size of from about 50 nm toabout 150 nm, more typically about 100 nm to about 130 nm, mosttypically about 110 nm to about 115 nm. The particles thus formed do notaggregate and are optionally sized to achieve a uniform particle size.

In some embodiments, the particles are formed using detergent dialysis.Without intending to be bound by any particular mechanism of formation,a plasmid or other nucleic acid (e.g., siRNA) is contacted with adetergent solution of cationic lipids to form a coated nucleic acidcomplex. These coated nucleic acids can aggregate and precipitate.However, the presence of a detergent reduces this aggregation and allowsthe coated nucleic acids to react with excess lipids (typically,non-cationic lipids) to form particles in which the plasmid or othernucleic acid is encapsulated in a lipid bilayer. Thus, the presentinvention provides a method for the preparation of serum-stable nucleicacid-lipid particles, comprising:

-   -   (a) combining a nucleic acid with cationic lipids in a detergent        solution to form a coated nucleic acid-lipid complex;    -   (b) contacting non-cationic lipids with the coated nucleic        acid-lipid complex to form a detergent solution comprising a        nucleic acid-lipid complex and non-cationic lipids; and    -   (c) dialyzing the detergent solution of step (b) to provide a        solution of serum-stable nucleic acid-lipid particles, wherein        the nucleic acid is encapsulated in a lipid bilayer and the        particles are serum-stable and have a size of from about 50 to        about 150 nm.

An initial solution of coated nucleic acid-lipid complexes is formed bycombining the nucleic acid with the cationic lipids in a detergentsolution.

In these embodiments, the detergent solution is preferably an aqueoussolution of a neutral detergent having a critical micelle concentrationof 15-300 mM, more preferably 20-50 mM. Examples of suitable detergentsinclude, for example,N,N′-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide) (BIGCHAP);BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether; Tween 20;Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9; Zwittergent®3-08; Zwittergent® 3-10; Triton X-405; hexyl-, heptyl-, octyl- andnonyl-β-D-glucopyranoside; and heptylthioglucopyranoside; with octylβ-D-glucopyranoside and Tween-20 being the most preferred. Theconcentration of detergent in the detergent solution is typically about100 mM to about 2 M, preferably from about 200 mM to about 1.5 M.

The cationic lipids and nucleic acids will typically be combined toproduce a charge ratio (+/−) of about 1:1 to about 20:1, preferably in aratio of about 1:1 to about 12:1, and more preferably in a ratio ofabout 2:1 to about 6:1. Additionally, the overall concentration ofnucleic acid in solution will typically be from about 25 μg/mL to about1 mg/mL, preferably from about 25 μg/mL to about 200 μm/mL, and morepreferably from about 50 μg/mL to about 100 μm/mL. The combination ofnucleic acids and cationic lipids in detergent solution is kept,typically at room temperature, for a period of time which is sufficientfor the coated complexes to form. Alternatively, the nucleic acids andcationic lipids can be combined in the detergent solution and warmed totemperatures of up to about 37° C. For nucleic acids which areparticularly sensitive to temperature, the coated complexes can beformed at lower temperatures, typically down to about 4° C.

In a preferred embodiment, the nucleic acid to lipid ratios (mass/massratios) in a formed nucleic acid-lipid particle will range from about0.01 to about 0.08. The ratio of the starting materials also fallswithin this range because the purification step typically removes theunencapsulated nucleic acid as well as the empty liposomes. In anotherpreferred embodiment, the nucleic acid-lipid particle preparation usesabout 400 μg nucleic acid per 10 mg total lipid or a nucleic acid tolipid ratio of about 0.01 to about 0.08 and, more preferably, about0.04, which corresponds to 1.25 mg of total lipid per 50 μg of nucleicacid.

The detergent solution of the coated nucleic acid-lipid complexes isthen contacted with non-cationic lipids to provide a detergent solutionof nucleic acid-lipid complexes and non-cationic lipids. Thenon-cationic lipids which are useful in this step include,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cardiolipin, and cerebrosides. In preferredembodiments, the non-cationic lipids are diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide or sphingomyelin. The acylgroups in these lipids are preferably acyl groups derived from fattyacids having C₁₀-C₂₄ carbon chains. More preferably the acyl groups arelauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In particularlypreferred embodiments, the non-cationic lipid will be1,2-sn-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC),distearoylphosphatidylcholine (DSPC), cholesterol, or a mixture thereof.In the most preferred embodiments, the nucleic acid-lipid particles willbe fusogenic particles with enhanced properties in vivo and thenon-cationic lipid will be DSPC or DOPE. In addition, the nucleicacid-lipid particles of the present invention may further comprisecholesterol. In other preferred embodiments, the non-cationic lipidswill further comprise polyethylene glycol-based polymers such as PEG2000, PEG 5000 and polyethylene glycol conjugated to a diacylglycerol, aceramide or a phospholipid, as described in U.S. Pat. No. 5,820,873 andU.S. Patent Publication No. 20030077829. In further preferredembodiments, the non-cationic lipids will further comprise polyethyleneglycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycolconjugated to a dialkyloxypropyl.

The amount of non-cationic lipid which is used in the present methods istypically about 2 to about 20 mg of total lipids to 50 μg of nucleicacid. Preferably the amount of total lipid is from about 5 to about 10mg per 50 μg of nucleic acid.

Following formation of the detergent solution of nucleic acid-lipidcomplexes and non-cationic lipids, the detergent is removed, preferablyby dialysis. The removal of the detergent results in the formation of alipid-bilayer which surrounds the nucleic acid providing serum-stablenucleic acid-lipid particles which have a size of from about 50 nm toabout 150 nm, more typically about 100 nm to about 130 nm, mosttypically about 110 nm to about 115 nm. The particles thus formed do notaggregate and are optionally sized to achieve a uniform particle size.

The serum-stable nucleic acid-lipid particles can be sized by any of themethods available for sizing liposomes. The sizing may be conducted inorder to achieve a desired size range and relatively narrow distributionof particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles is described in U.S. Pat. No. 4,737,323.Sonicating a particle suspension either by bath or probe sonicationproduces a progressive size reduction down to particles of less thanabout 50 nm in size. Homogenization is another method which relies onshearing energy to fragment larger particles into smaller ones. In atypical homogenization procedure, particles are recirculated through astandard emulsion homogenizer until selected particle sizes, typicallybetween about 60 and 80 nm, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In another group of embodiments, the present invention provides a methodfor the preparation of serum-stable nucleic acid-lipid particles,comprising:

-   -   (a) preparing a mixture comprising cationic lipids and        non-cationic lipids in an organic solvent;    -   (b) contacting an aqueous solution of nucleic acid with said        mixture in step (a) to provide a clear single phase; and    -   (c) removing said organic solvent to provide a suspension of        nucleic acid-lipid particles, wherein said nucleic acid is        encapsulated in a lipid bilayer, and said particles are stable        in serum and have a size of from about 50 to about 150 nm.

The nucleic acids (or plasmids), cationic lipids and non-cationic lipidswhich are useful in this group of embodiments are as described for thedetergent dialysis methods above.

The selection of an organic solvent will typically involve considerationof solvent polarity and the ease with which the solvent can be removedat the later stages of particle formation. The organic solvent, which isalso used as a solubilizing agent, is in an amount sufficient to providea clear single phase mixture of nucleic acid and lipids. Suitablesolvents include, but are not limited to, chloroform, dichloromethane,diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, orother aliphatic alcohols such as propanol, isopropanol, butanol,tert-butanol, iso-butanol, pentanol and hexanol. Combinations of two ormore solvents may also be used in the present invention.

Contacting the nucleic acid with the organic solution of cationic andnon-cationic lipids is accomplished by mixing together a first solutionof nucleic acid, which is typically an aqueous solution, and a secondorganic solution of the lipids. One of skill in the art will understandthat this mixing can take place by any number of methods, for example bymechanical means such as by using vortex mixers.

After the nucleic acid has been contacted with the organic solution oflipids, the organic solvent is removed, thus forming an aqueoussuspension of serum-stable nucleic acid-lipid particles. The methodsused to remove the organic solvent will typically involve evaporation atreduced pressures or blowing a stream of inert gas (e.g., nitrogen orargon) across the mixture.

The serum-stable nucleic acid-lipid particles thus formed will typicallybe sized from about 50 nm to about 150 nm, more typically about 100 nmto about 130 nm, most typically about 110 nm to about 115 nm. To achievefurther size reduction or homogeneity of size in the particles, sizingcan be conducted as described above.

In other embodiments, the methods will further comprise adding nonlipidpolycations which are useful to effect the delivery to cells using thepresent compositions. Examples of suitable nonlipid polycations include,but are limited to, hexadimethrine bromide (sold under the brandnamePOLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or othersalts of heaxadimethrine. Other suitable polycations include, forexample, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine,poly-D-lysine, polyallylamine and polyethyleneimine.

In certain embodiments, the formation of the nucleic acid-lipidparticles can be carried out either in a mono-phase system (e.g., aBligh and Dyer monophase or similar mixture of aqueous and organicsolvents) or in a two-phase system with suitable mixing.

When formation of the complexes is carried out in a mono-phase system,the cationic lipids and nucleic acids are each dissolved in a volume ofthe mono-phase mixture. Combination of the two solutions provides asingle mixture in which the complexes form. Alternatively, the complexescan form in two-phase mixtures in which the cationic lipids bind to thenucleic acid (which is present in the aqueous phase), and “pull” it intothe organic phase.

In another embodiment, the present invention provides a method for thepreparation of nucleic acid-lipid particles, comprising:

-   -   (a) contacting nucleic acids with a solution comprising        non-cationic lipids and a detergent to form a nucleic acid-lipid        mixture;    -   (b) contacting cationic lipids with the nucleic acid-lipid        mixture to neutralize a portion of the negative charge of the        nucleic acids and form a charge-neutralized mixture of nucleic        acids and lipids; and    -   (c) removing the detergent from the charge-neutralized mixture        to provide the nucleic acid-lipid particles in which the nucleic        acids are protected from degradation.

In one group of embodiments, the solution of non-cationic lipids anddetergent is an aqueous solution. Contacting the nucleic acids with thesolution of non-cationic lipids and detergent is typically accomplishedby mixing together a first solution of nucleic acids and a secondsolution of the lipids and detergent. One of skill in the art willunderstand that this mixing can take place by any number of methods, forexample, by mechanical means such as by using vortex mixers. Preferably,the nucleic acid solution is also a detergent solution. The amount ofnon-cationic lipid which is used in the present method is typicallydetermined based on the amount of cationic lipid used, and is typicallyof from about 0.2 to 5 times the amount of cationic lipid, preferablyfrom about 0.5 to about 2 times the amount of cationic lipid used.

In some embodiments, the nucleic acids are precondensed as described in,e.g., U.S. patent application Ser. No. 09/744,103.

The nucleic acid-lipid mixture thus formed is contacted with cationiclipids to neutralize a portion of the negative charge which isassociated with the nucleic acids (or other polyanionic materials)present. The amount of cationic lipids used will typically be sufficientto neutralize at least 50% of the negative charge of the nucleic acid.Preferably, the negative charge will be at least 70% neutralized, morepreferably at least 90% neutralized. Cationic lipids which are useful inthe present invention, include, for example, DLinDMA and, DLenDMA. Theselipids and related analogs have been described in U.S. ProvisionalPatent Application Nos. 60/578,075, filed Jun. 7, 2004; 60/610,746,filed Sep. 17, 2004; and 60/679,427, filed May 9, 2005.

Contacting the cationic lipids with the nucleic acid-lipid mixture canbe accomplished by any of a number of techniques, preferably by mixingtogether a solution of the cationic lipid and a solution containing thenucleic acid-lipid mixture. Upon mixing the two solutions (or contactingin any other manner), a portion of the negative charge associated withthe nucleic acid is neutralized. Nevertheless, the nucleic acid remainsin an uncondensed state and acquires hydrophilic characteristics.

After the cationic lipids have been contacted with the nucleicacid-lipid mixture, the detergent (or combination of detergent andorganic solvent) is removed, thus forming the nucleic acid-lipidparticles. The methods used to remove the detergent will typicallyinvolve dialysis. When organic solvents are present, removal istypically accomplished by evaporation at reduced pressures or by blowinga stream of inert gas (e.g., nitrogen or argon) across the mixture.

The particles thus formed will typically be sized from about 50 nm toseveral microns, more typically about 50 nm to about 150 nm, even moretypically about 100 nm to about 130 nm, most typically about 110 nm toabout 115 nm. To achieve further size reduction or homogeneity of sizein the particles, the nucleic acid-lipid particles can be sonicated,filtered or subjected to other sizing techniques which are used inliposomal formulations and are known to those of skill in the art.

In other embodiments, the methods will further comprise adding nonlipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable nonlipid polycationsinclude, hexadimethrine bromide (sold under the brandname POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In another aspect, the present invention provides methods for thepreparation of nucleic acid-lipid particles, comprising:

-   -   (a) contacting an amount of cationic lipids with nucleic acids        in a solution; the solution comprising from about 15-35% water        and about 65-85% organic solvent and the amount of cationic        lipids being sufficient to produce a +/− charge ratio of from        about 0.85 to about 2.0, to provide a hydrophobic nucleic        acid-lipid complex;    -   (b) contacting the hydrophobic, nucleic acid-lipid complex in        solution with non-cationic lipids, to provide a nucleic        acid-lipid mixture; and    -   (c) removing the organic solvents from the nucleic acid-lipid        mixture to provide nucleic acid-lipid particles in which the        nucleic acids are protected from degradation.

The nucleic acids, non-cationic lipids, cationic lipids and organicsolvents which are useful in this aspect of the invention are the sameas those described for the methods above which used detergents. In onegroup of embodiments, the solution of step (a) is a mono-phase. Inanother group of embodiments, the solution of step (a) is two-phase.

In preferred embodiments, the non-cationic lipids are ESM, DOPE, DOPC,polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000,PEG-modified diacylglycerols, or PEG-modified dialkyloxypropyls),distearoylphosphatidylcholine (DSPC), DPPE, DMPE, 16:0 MonomethylPhosphatidylethanolamine, 16:0 Dimethyl Phosphatidylethanolamine, 18:1

Trans Phosphatidylethanolamine, 18:0 18:1 Phosphatidylethanolamine(SOPE), 16:0 18:1 Phosphatidylethanolamine, DSPE, cholesterol, orcombinations thereof. In still other preferred embodiments, the organicsolvents are methanol, chloroform, methylene chloride, ethanol, diethylether or combinations thereof

In one embodiment, the nucleic acid is a plasmid from which aninterfering RNA is transcribed; the cationic lipid is DLindMA, DLenDMA,DODAC, DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations thereof; thenon-cationic lipid is ESM, DOPE, DAG-PEGs, distearoylphosphatidylcholine(DSPC), DPPE, DMPE, 16:0 Monomethyl Phosphatidylethanolamine, 16:0Dimethyl Phosphatidylethanolamine, 18:1 Trans Phosphatidylethanolamine,18:0 18:1 Phosphatidylethanolamine (SOPE), 16:0 18:1Phosphatidylethanolamine DSPE, cholesterol, or combinations thereof(e.g. DSPC and PEG-DAA); and the organic solvent is methanol,chloroform, methylene chloride, ethanol, diethyl ether or combinationsthereof

As above, contacting the nucleic acids with the cationic lipids istypically accomplished by mixing together a first solution of nucleicacids and a second solution of the lipids, preferably by mechanicalmeans such as by using vortex mixers. The resulting mixture containscomplexes as described above. These complexes are then converted toparticles by the addition of non-cationic lipids and the removal of theorganic solvent. The addition of the non-cationic lipids is typicallyaccomplished by simply adding a solution of the non-cationic lipids tothe mixture containing the complexes. A reverse addition can also beused. Subsequent removal of organic solvents can be accomplished bymethods known to those of skill in the art and also described above.

The amount of non-cationic lipids which is used in this aspect of theinvention is typically an amount of from about 0.2 to about 15 times theamount (on a mole basis) of cationic lipids which was used to providethe charge-neutralized nucleic acid-lipid complex. Preferably, theamount is from about 0.5 to about 9 times the amount of cationic lipidsused.

In yet another aspect, the present invention provides nucleic acid-lipidparticles which are prepared by the methods described above. In theseembodiments, the nucleic acid-lipid particles are either net chargeneutral or carry an overall charge which provides the particles withgreater gene lipofection activity. Preferably, the nucleic acidcomponent of the particles is a nucleic acid which interferes with theproduction of an undesired protein. In a preferred embodiment, thenucleic acid comprises an interfering RNA, the non-cationic lipid is eggsphingomyelin and the cationic lipid is DLinDMA or DLenDMA. In apreferred embodiment, the nucleic acid comprises an interfering RNA, thenon-cationic lipid is a mixture of DSPC and cholesterol, and thecationic lipid is DLinDMA or DLenDMA. In other preferred embodiments,the non-cationic lipid may further comprise cholesterol.

A variety of general methods for making SNALP-CPLs (CPL-containingSNALPs) are discussed herein. Two general techniques include“post-insertion” technique, that is, insertion of a CPL into forexample, a pre-formed SNALP, and the “standard” technique, wherein theCPL is included in the lipid mixture during for example, the SNALPformation steps. The post-insertion technique results in SNALPs havingCPLs mainly in the external face of the SNALP bilayer membrane, whereasstandard techniques provide SNALPs having CPLs on both internal andexternal faces. The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of makingSNALP-CPL, are taught, for example, in U.S. Pat. Nos. 5,705,385,6,586,410, 5,981,501 6,534,484; 6,852,334; U.S. Patent Publication No.20020072121; and WO 00/62813.

IV. Administration of Nucleic Acid-Lipid Particle Formulations

Once formed, the serum-stable nucleic acid-lipid particles of thepresent invention are useful for the introduction of nucleic acids intocells. Accordingly, the present invention also provides methods forintroducing a nucleic acids (e.g., a plasmid or and siRNA) into a cell.The methods are carried out in vitro or in vivo by first forming theparticles as described above and then contacting the particles with thecells for a period of time sufficient for delivery of the nucleic acidto the cell to occur.

The nucleic acid-lipid particles of the present invention can beadsorbed to almost any cell type with which they are mixed or contacted.Once adsorbed, the particles can either be endocytosed by a portion ofthe cells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the nucleic acid portion of the particlecan take place via any one of these pathways. In particular, when fusiontakes place, the particle membrane is integrated into the cell membraneand the contents of the particle combine with the intracellular fluid.

The nucleic acid-lipid particles of the present invention can beadministered either alone or in mixture with aphysiologically-acceptable carrier (such as physiological saline orphosphate buffer) selected in accordance with the route ofadministration and standard pharmaceutical practice. Generally, normalsaline will be employed as the pharmaceutically acceptable carrier.Other suitable carriers include, e.g., water, buffered water, 0.4%saline, 0.3% glycine, and the like, including glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc.

The pharmaceutical carrier is generally added following particleformation. Thus, after the particle is formed, the particle can bediluted into pharmaceutically acceptable carriers such as normal saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2-5% to as much as 10 to 30% by weight and will be selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The nucleic acid-lipid particles can be incorporated into a broad rangeof topical dosage forms including, but not limited to, gels, oils,emulsions, topical creams, pastes, ointments, lotions and the like.

A. In Vivo Administration

Systemic delivery for in vivo gene therapy, i.e., delivery of atherapeutic nucleic acid to a distal target cell via body systems suchas the circulation, has been achieved using nucleic acid-lipid particlessuch as those disclosed in WO 96/40964, U.S. Pat. Nos. 5,705,385,5,976,567, 5,981,501, and 6,410,328. This latter format provides a fullyencapsulated nucleic acid-lipid particle that protects the nucleic acidfrom nuclease degradation in serum, is nonimmunogenic, is small in sizeand is suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation,transdermal application, or rectal administration. Administration can beaccomplished via single or divided doses. The pharmaceuticalcompositions are preferably administered parenterally, i.e.,intraarticularly, intravenously, intraperitoneally, subcutaneously, orintramuscularly. More preferably, the pharmaceutical compositions areadministered intravenously or intraperitoneally by a bolus injection(see, e.g., Stadler, et al., U.S. Pat. No. 5,286,634). Intracellularnucleic acid delivery has also been discussed in Straubringer, et al.,Methods Enzymol, Academic Press, New York. 101:512 (1983); Mannino, etal., Biotechniques 6:682 (1988); Nicolau, et al., Crit. Rev. Ther. DrugCarrier Syst. 6:239 (1989), and Behr, Acc. Chem. Res. 26:274 (1993).Still other methods of administering lipid based therapeutics aredescribed in, for example, Rahman et al., U.S. Pat. No. 3,993,754;Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No.4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat.No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. The lipidnucleic acid particles can be administered by direct injection at thesite of disease or by injection at a site distal from the site ofdisease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc.,Publishers, New York. pp. 70-71(1994)).

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (see, Brigham, et al., Am. J. Sci. 298(4):278 (1989)).Aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

Generally, when administered intravenously, the nucleic acid-lipidformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

When preparing pharmaceutical preparations of the nucleic acid-lipidparticles of the invention, it is preferable to use quantities of theparticles which have been purified to reduce or eliminate emptyparticles or particles with nucleic acid associated with the externalsurface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as avian (e.g.,ducks), primates (e.g., humans and chimpanzees as well as other nonhumanprimates), canines, felines, equines, bovines, ovines, caprines, rodents(e.g., rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio ofnucleic acid to lipid; the particular nucleic acid used, the diseasestate being diagnosed; the age, weight, and condition of the patient andthe judgment of the clinician; but will generally be between about 0.01and about 50 mg per kilogram of body weight; preferably between about0.1 and about 5 mg/kg of body weight or about 10⁸-10¹⁰ particles perinjection.

B. Cells for Delivery of Interfering RNA

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, e.g., hematopoietic precursor (stem) cells, fibroblasts,keratinocytes, hepatocytes, endothelial cells, skeletal and smoothmuscle cells, osteoblasts, neurons, quiescent lymphocytes, terminallydifferentiated cells, slow or noncycling primary cells, parenchymalcells, lymphoid cells, epithelial cells, bone cells, and the like.

In vivo delivery of nucleic acid lipid particles encapsulating aninterfering RNA is particularly suited for targeting tumor cells of anycell type. In vivo studies show that SNALP's accumulate at tumor sitesand predominantly transfect tumor cells. See, Fenske, et al., MethodsEnzymol, Academic Press, New York 346:36 (2002). The methods andcompositions can be employed with cells of a wide variety ofvertebrates, including mammals, and especially those of veterinaryimportance, e.g., canine, feline, equine, bovine, ovine, caprine,rodent, lagomorph, swine, etc., in addition to human cell populations.

To the extent that tissue culture of cells may be required, it is wellknown in the art. Freshney (1994) (Culture of Animal Cells, a Manual ofBasic Technique, third edition Wiley-Liss, New York), Kuchler et al.(1977) Biochemical Methods in Cell Culture and Virology, Kuchler, R. J.,Dowden, Hutchinson and Ross, Inc., and the references cited thereinprovides a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

C. Detection of SNALPs

In some embodiments, the nucleic acid-lipid particles are detectable inthe subject 8, 12, 24, 48, 60, 72, or 96 hours after administration ofthe particles. The presence of the particles can be detected in thecells, tissues, or other biological samples from the subject. Theparticles by be detected, e.g., by direct detection of the particles,detection of the interfering RNA sequence, detection of the targetsequence of interest (i.e., by detecting expression or reducedexpression of the sequence of interest), or a combination thereof.

1. Detection of Particles

Nucleic acid-lipid particles are detected herein using any methods knownin the art. For example, a label can be coupled directly or indirectlyto a component of the SNALP or other lipid-based carrier system usingmethods well known in the art. A wide variety of labels can be used,with the choice of label depending on sensitivity required, ease ofconjugation with the SNALP component, stability requirements, andavailable instrumentation and disposal provisions. Suitable labelsinclude, but are not limited to, spectral labels, such as fluorescentdyes (e.g., fluorescein and derivatives, such as fluoresceinisothiocyanate (FITC) and Oregon Green™; rhodamine and derivatives, suchTexas red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin,biotin, phycoerythrin, AMCA, CyDyes™, and the like; radiolabels, such as³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes, such as horse radishperoxidase, alkaline phosphatase, etc.; spectral colorimetric labels,such as colloidal gold or colored glass or plastic beads, such aspolystyrene, polypropylene, latex, etc. The label can be detected usingany means known in the art.

2. Detection of Nucleic Acids

Nucleic acids are detected and quantified herein by any of a number ofmeans well known to those of skill in the art. The detection of nucleicacids proceeds by well known methods such as Southern analysis, northernanalysis, gel electrophoresis, PCR, radiolabeling, scintillationcounting, and affinity chromatography. Additional analytic biochemicalmethods such as spectrophotometry, radiography, electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,may also be employed

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in “Nucleic Acid Hybridization, A PracticalApproach,” Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR) theligase chain reaction (LCR), Qβ-replicase amplification and other RNApolymerase mediated techniques (e.g., NASBA™) are found in Sambrook, etal., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, 2000, and Ausubel et al., SHORT PROTOCOLS IN MOLECULARBIOLOGY, eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (2002), as wellas Mullis et al. (1987), U.S. Pat. No. 4,683,202; PCR Protocols A Guideto Methods and Applications (Innis et al. eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990), C&EN36; The Journal Of NIH Research, 3:81 (1991); (Kwoh et al., Proc. Natl.Acad. Sci. USA, 86:1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci.USA, 87:1874 (1990); Lomell et al., J. Clin. Chem., 35:1826 (1989);Landegren et al., Science, 241:1077 (1988); Van Brunt, Biotechnology,8:291 (1990); Wu and Wallace, Gene, 4:560 (1989); Barringer et al.,Gene, 89:117 (1990), and Sooknanan and Malek, Biotechnology, 13:563(1995). Improved methods of cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039. Other methodsdescribed in the art are the nucleic acid sequence based amplification(NASBA™, Cangene, Mississauga, Ontario) and Q Beta Replicase systems.These systems can be used to directly identify mutants where the PCR orLCR primers are designed to be extended or ligated only when a selectsequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation.

Oligonucleotides for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts., 22(20):1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of oligonucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson and Regnier, J.Chrom., 255:137 149 (1983). The sequence of the syntheticoligonucleotides can be verified using the chemical degradation methodof Maxam and Gilbert (1980) in Grossman and Moldave (eds.) AcademicPress, New York, Methods in Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

D. Transfection Efficiency

The transfection efficiency of the nucleic acid-lipid particlesdescribed herein can be optimized using an ERP assay. For example, theERP assay can be used to distinguish the effect of various cationiclipids, non-cationic lipids, and bilayer stabilizing components of theSNALPs based on their relative effect on binding/uptake or fusionwith/destabilization of the endosomal membrane. This assay allows one todetermine quantitatively how each component of the SNALPs affectstransfection efficacy, thereby optimizing the SNALPs. As explainedherein, the Endosomal Release Parameter or, alternatively, ERP isdefined as:

Reporter Gene Expression/Cell

SNALP Uptake/Cell

It will be readily apparent to those of skill in the art that anyreporter gene (e.g., luciferase, β-galactosidase, green fluorescentprotein, etc.) can be used. In addition, the lipid component (or,alternatively, any component of the SNALP or lipid-based formulation)can be labeled with any detectable label provided the does inhibit orinterfere with uptake into the cell. Using the ERP assay of the presentinvention, one of skill in the art can assess the impact of the variouslipid components (e.g., cationic lipid of Formula I or II, non-cationiclipid, PEG-lipid derivative, PEG-DAA conjugate, ATTA-lipid derivative,calcium, CPLs, cholesterol, etc.) on cell uptake and transfectionefficiencies, thereby optimizing the SPLP or other lipid-based carriersystem. By comparing the ERPs for each of the various SPLPs or otherlipid-based formulations, one can readily determine the optimizedsystem, e.g., the SPLP or other lipid-based formulation that has thegreatest uptake in the cell coupled with the greatest transfectionefficiency.

Suitable labels for carrying out the ERP assay of the present inventioninclude, but are not limited to, spectral labels, such as fluorescentdyes (e.g., fluorescein and derivatives, such as fluoresceinisothiocyanate (FITC) and Oregon Green; rhodamine and derivatives, suchTexas red, tetrarhodimine isothiocynate (TRITC), etc., digoxigenin,biotin, phycoerythrin, AMCA, CyDyes^(θ), and the like; radiolabels, suchas ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes, such as horse radishperoxidase, alkaline phosphatase, etc.; spectral colorimetric labels,such as colloidal gold or colored glass or plastic beads, such aspolystyrene, polypropylene, latex, etc. The label can be coupleddirectly or indirectly to a component of the SNALP using methods wellknown in the art. As indicated above, a wide variety of labels can beused, with the choice of label depending on sensitivity required, easeof conjugation with the SNALP component, stability requirements, andavailable instrumentation and disposal provisions.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Materials and Methods

Materials:

DPPS, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterolwere purchased from Avanti Polar Lipids (Alabaster, Ala.). TNS wasobtained from Sigma-Aldrich Canada (Oakville, ON). RiboGreen wasobtained from Molecular Probes (Eugene, Oreg.). The alkyl mesylates werepurchased from Nu-Chek Prep, Inc. (Elysian, Minn., USA). siRNA(anti-luciferase and mismatch control) was purchased from Dharmacon(Lafayette, Colo., USA). The anti-luciferase sense sequence was5′-GAUUAUGUCCGGUUAUGUAUU-3′ (SEQ ID NO:1). The anti-luciferase antisensesequence was 5′-UACAUAACCGGACAUAAUCUU-3′ (SEQ ID NO:2). All otherchemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Synthesis of DSDMA and DODMA:

DSDMA and DODMA were synthesized using the respective alkyl bromideswith methodology derived from that of a DOTMA precursor (Feigner et al,PNAS USA, 84, 7413-7417 (1987)). 3-(Dimethylamino)-1,2-propanediol (714mg, 6 mmol) and 95% sodium hydride (NaH, 1.26 g, 50 mmol) were stirredin benzene (30 mL) under argon for 30 minutes. The correct (either oleylor stearyl) alkyl bromide (5.0 g, 15 mmol) was added and the reactionrefluxed under argon for 18 hours. The reaction mixture was then cooledin an ice bath while quenching via the slow addition of ethanol.Following dilution with a further 150 mL of benzene, the mixture waswashed with distilled water (2×150 mL) and brine (150 mL), using ethanol(˜20 mL) to aid phase separation if necessary. The organic phase wasdried over magnesium sulphate and evaporated. The crude product waspurified on a silica gel (Kiesel Gel 60) column eluted with chloroformcontaining 0-5% methanol. Column fractions were analyzed by thin layerchromatography (TLC) (silica gel, chloroform/methanol 9:1 v/v,visualized with molybdate) and fractions containing pure product(R_(f)=0.5) were pooled and concentrated. The product was decolorized bystirring for 30 minutes in a suspension of activated charcoal (1 g) inethanol (75 mL) at 60° C. The charcoal was removed by filtration throughCelite, and the ethanol solution concentrated to typically yield 2.4 g(65%) of pure product. ¹H-NMR (DSDMA): δ_(H) 3.65-3.32 (m, 7H, OCH,3×OCH₂), 2.45-2.31 (m, 2H, NCH₂), 2.27 (s, 6H, 2×NCH₃), 1.61-1.45 (m,4H, OCH₂ CH ₂), 1.40-1.17 (m, 60H, H_(stearyl)), 0.86 (t, 6H, CH₂CH ₃).¹H-NMR (DODMA): δ_(H) 5.4-5.27 (m, 4H, 2×CH═CH), 3.65-3.35 (m, 7H, OCH,3×OCH₂), 2.47-2.33 (m, 2H, NCH₂), 2.28 (s, 6H, 2×NCH₃), 2.06-1.94 (m,8H, 4×CH ₂CH═CH), 1.61-1.50 (m, 4H, OCH₂CH ₂), 1.38-1.20 (m, 48H,H_(oleyl)), 0.88 (t, 6H, CH₂CH ₃).

Synthesis of 1, 2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA) and1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA)

3-(Dimethylamino)-1,2-propanediol (714 mg, 6 mmol) and 95% sodiumhydride (NaH, 1.26 g, 50 mmol) are stirred in benzene (30 mL) undernitrogen for 30 minutes. Linoleyl mesylate (5.0 g, 15 mmol) is added andthe reaction refluxed under nitrogen for 3 hours. The reaction mixtureis then cooled in an ice bath while quenching via the slow addition ofethanol. Following dilution with a further 150 mL of benzene, themixture is washed with distilled water (2×150 mL) and brine (150 mL).The organic phase is dried over magnesium sulphate and evaporated togive the crude product.

The crude product is purified on a silica gel (Kiesel Gel 60) columneluted with 0-5% methanol in chloroform. Column fractions are analyzedby thin layer chromatography (TLC) (silica gel, chloroform/methanol 9:1v/v, visualized with molybdate dip) and fractions containing purifiedproduct (R_(f)=0.5) are pooled and concentrated.

Decolorization and further purification of DLinDMA is effected with asecond column, this time eluting with 20-50% ethyl acetate in hexane.Column fractions are analyzed by TLC (silica gel, ethyl acetate/hexane1:1 v/v, visualized with molybdate) and fractions containing pureproduct (R_(f)=0.4) are pooled and concentrated. The procedure describedherein typically yields 2.2 g (60%) of pure product.

For synthesis of DLenDMA, linolenyl mesylate is substituted for linoleylmesylate and the remainder of the synthesis, decolorization, andpurification reactions is carried out as described above.

Synthesis of PEG₂₀₀₀-C-DMA:

PEG-C-DMA was synthesized as follows. In brief, a C₁₄ lipid anchor wasprepared by first alkylating the hydroxyl groups of3-allyloxypropane-1,2-diol with myristyl bromide. The allyl group wassubsequently removed via palladium catalysis, resulting in the C₁₄hydroxyl lipid. The hydroxyl group was converted to the primary amine bymesylation and amination to yield 1,2-dimyristyloxypropyl-3-amine, thelipid anchor. Conjugation with PEG was effected by treating monomethoxypoly(ethylene glycol) (average molecular weight 2000) with an excess ofdiphosgene to form the chloroformate. Addition of the C₁₄ amine lipidanchor and stirring overnight yielded PEG₂₀₀₀-C-DMA, referred to here asPEG-C-DMA.

SNALP Preparation:

SNALP with a lipid composition of DSPC:Chol:PEG-C-DMA:Cationic Lipid(20:48:2:30 molar percent) were prepared using the spontaneous vesicleformation by ethanol dilution method [Jeffs et al., Pharm. Res. In Press(2005)]. The sample's were diafiltered against 100 mL of PBS (20 washvolumes) using a cross flow ultrafiltration cartridge (AmershamBiosciences, Piscataway, N.J.) and sterile filtered through Acrodisc 0.2μm Posidyne filters (Pall Corp., Ann Arbor, Mich.). The siRNAconcentration of final samples was determined using the RiboGreen assayand a siRNA standard curve. Particle size and polydispersity wasdetermined using a Malvern Instruments Zetasizer 3000HSA (Malvern, UK).Nucleic acid encapsulation was determined using a RiboGreen assay,comparing fluorescence in the presence and absence of Triton X-100.RiboGreen fluorescence was measured using a Varian EclipseSpectrofluorometer (Varian Inc) with λ_(ex)=500 nm, λ_(em)=525 nm.

TNS Assay:

20 μM of SNALP lipid and 6 μM of TNS were mixed in a fluorescencecuvette in 2 mL of 20 mM sodium phosphate, 25 mM citrate, 20 mM ammoniumacetate and 150 mM NaCl, at a pH that was varied from 4.5 to 9.5.Fluorescence was determined at each pH using a Varian EclipseSpectrofluorometer (Varian Inc) with settings of λ_(ex)=322 nm,λ_(em)=431 nm. Fluorescence for each system at the various pH was thennormalized to the value at pH 4.5. The pK_(a) values are the point atwhich 50% of the molecules present are charged. By assuming that minimumfluorescence represents zero charge, and maximum fluorescence represents100% charge, pK_(a) can be estimated by measuring the pH at the pointexactly half way between the values of minimum and maximum charge.

³¹P Nuclear Magnetic Resonance Spectroscopy:

Multilamellar vesicles (MLV) were prepared comprising DPPS and cationiclipid at a molar ratio of 1:1. This was accomplished by drying thelipids from chloroform solution, transferring to 10 mm NMR tubes, andhydrating in 1.5 mL of 10 mM sodium citrate, pH 4. Free induction decays(FIDs) corresponding to 1000 scans were obtained with a 3.0 μs, 60 opulse with a 1 s interpulse delay and a spectral width of 25000 Hz. Agated two-level proton decoupling was used to ensure sufficientdecoupling with minimum sample heating. An exponential multiplicationcorresponding to 50 Hz of line broadening was applied to the FIDs priorto Fourier transformation. The sample temperature (+/−1° C.) wasregulated using a Bruker B-VT1000 variable temperature unit. Chemicalshifts were referenced to 85% phosphoric acid as an external standard.

In Vitro Transfection:

Cells were cultured in MEM (Invitrogen) containing 10% fetal bovineserum (FBS) (CanSera) and 0.25 mg/mL G418 (Invitrogen). Neuro2A-G cells(Neuro2A cells stably transfected to express luciferase [R. E. Kingston.in Current Protocols in Molecular Biology, Vol. 2, pp. 9.1.4-9.1.9, JohnWiley & Sons, Inc. (1997)]) were plated at a concentration of 4×10⁴cells per well in 24-well plates and grown overnight. Cells were treatedwith SNALP at doses of 0.0625-1.0 μg/mL nucleic acid (AntiLuc Active orMismatch Control) and incubated for 48 hours at 37° C. and 5% CO₂. Cellswere then washed with PBS and lysed with 200 μL 250 mM sodium phosphatecontaining 0.1% Triton X-100. The luciferase activity for each well wasdetermined using Luciferase Reagent (Promega) and a standard luciferaseprotein (Roche). The luminescence for each was measured using a BertholdMicroLumatPlus LB96V plate luminometer. The resulting luciferaseactivity was then normalized for the amount of protein using the MicroBCA assay kit (Pierce). Luciferase knockdown relative to a control wasthen determined for each system.

Cellular Uptake:

SNALP were prepared incorporating the non-exchangeable tritium-labeledlipid cholesteryl hexadecyl ether (3H—CHE) (11.1 μCi/μmol total lipid)[Bally et al., in Liposome Technology, Vol. III, pp. 27-41, CRC Press(1993)]. Neuro2A cells (ATCC, VA, USA) were plated in 12 well plates at1.6×105 cells per well in minimal essential media. The following day,media was removed and replaced with media containing radiolabelled SNALPat 0.5 m/mL nucleic acid. After 24 hours, the media and unincorporatedSNALP were removed, adherent cells gently washed 4 times with PBS, andthen lysed with 600 μL Lysis Buffer (250 mM phosphate with 0.1% TritonX-100). The resulting cell lysate (500 μL) was added to glassscintillation vials containing 5 mL Picofluor 40 (Perkin Elmer) and³H—CHE was determined using a Beckman LS6500 scintillation counter(Beckman Instruments). The protein content of cell lysates wasdetermined using the Micro BCA assay (Pierce). Uptake was expressed as apercentage of the total amount of activity applied to the cells per mgof cellular protein.

Uptake of SNALP Containing Cy3-Labeled siRNA:

SNALP were Formulated as previously described, but using siRNA labelledwith the fluorophore Cy3 (Cy3-siRNA was a gift of Sirna TherapeuticsInc, Boulder, Colo.). The encapsulation, siRNA concentration, andparticle size were determined as described.

For the uptake study, 8×10⁴ Neuro2A-G cells were grown overnight on4-well chamber slides (B D Falcon, Mississauga, ON) in MEM containing0.25 mg/mL G418. DSDMA, DODMA, DLinDMA, and DLenDMA SNALP containingCy3-siRNA, as well as naked Cy3-siRNA and unlabeled DSDMA SNALP wereplaced on the cells at 0.5 μg/mL siRNA. After a 4 hour incubation withthe transfection media, the cells were washed with PBS, then with MEMcontaining G418 and finally with PBS once more. The cells were thenfixed in a 4% paraformaldehyde solution in PBS for 10 min at roomtemperature. The cells were washed with PBS and stained with 300 nM DAPI(Molecular Probes, Eugene, Oreg.) in PBS for 5 minutes. The cells werewashed with PBS, the mounting media ProLong Gold Antifade Reagent(Molecular Probes, Eugene, Oreg.) applied and a cover slip added. Thecells were viewed using an Olympus BX60 Microscope modified forfluorescence capabilities. Cy3 fluorescence within the cells wasvisualized using a rhodamine cube set (Microgen Optics, Redding, Calif.)and the DAPI fluorescence was visualized using a DAPI cube set (CarsenGroup, Markham, ON). Digital pictures were captured using an OlympusDP70 camera system. Pictures of the cells were taken at exposure timesof ¼ sec when examining Cy3 fluorescence and 1/80 sec when examiningDAPI fluorescence.

Example 2 SNALP Formulations Encapsulating siRNA

This example demonstrates encapsulating siRNA in SNALP formulated witheither short- or long-chain PEG-DAG and produced by continuously mixingorganic lipid and aqueous buffer solutions. PEG-DAG lipids employed werePEG-dimyristylglycerol (C₁₄) (PEG-DMG) and PEG-distearylglycerol (C₁₈)(PEG-DSG). Anti-β-galactosidase (β-gal) siRNA encapsulated inDSPC:Cholesterol:DODMA:PEG-DMG/PEG-DSG SNALP by this method resulted in≥90% encapsulation (Ribogreen Assay) and ˜120 nm particle size (Malvernsizer). The preparations had the following characteristics:

4 ml prep: anti-B-gal siRNA in DSPC:Chol:DODMA:PEG-DMG liposomes

Initial mix=94% encapsulation

Post dilution mix=98% encapsulation

Post incubation mix=97% encapsulation

Post overnight dialysis=96% encapsulation

Particle size=109.7 nm

Polydispersity=0.14

8 ml prep: anti-B-gal siRNA in DSPC:Chol:DODMA:PEG-DMG liposomes

Post dilution & incubated mix=89%

Post overnight dialysis=91%

Particle size=127.5 nm

Polydispersity=0.11

8 ml prep: anti-B-gal siRNA in DSPC: Chol:DODMA:PEG-DSG liposomes

Post dilution & incubated mix=90%

Post overnight dialysis=90%

Post sterile-filter=90%

Particle size=111.6 nm

Polydispersity=0.24

Example 3 Downregulation of Intracellular Expression in Cells byDelivering In Vitro an SNALP Formulation Encapsulating siRNA

This example demonstrates downregulation of β-Gal expression inCT26.CL25 cells delivered in vitro DSPC:Cholesterol:DODMA:PEG-DMGliposomes encapsulating anti-β-Gal siRNA. The results are depicted inFIG. 4.

In vitro delivery of 0.2 μg Oligofectamine-encapsulated anti-β-Gal siRNAdecreased β-Gal activity by about 60% in comparison to unexposed controlcells. Encapsulating 1.5 μg anti-β-Gal siRNA inDSPC:Cholesterol:DODMA:PEG-DMG liposomes decreased β-Gal activity byabout 30% in comparison to unexposed control cells.

Example 4 Assays for Serum Stability

Lipid/therapeutic nucleic acid particles formulated according to theabove noted techniques can be assayed for serum stability by a varietyof methods.

For instance, in a typical DNase 1 digestion, 1 μg of DNA encapsulatedin the particle of interest is incubated in a total volume of 100 μL of5 mM HEPES, 150 mM NaCl, 10.0 mM MgCl₂ pH 7.4. DNase treated samples aretreated with either 100 or 10 U of DNase I (Gibco-BRL). 1.0% TritonX-100 can be added in control experiments to ensure that lipidformulations are not directly inactivating the enzyme. Samples areincubated at 37° C. for 30 min after which time the DNA is isolated byaddition of 500 μL of DNAZOL followed by 1.0 mL of ethanol. The samplesare centrifuged for 30 min at 15,000 rpm in a tabletop microfuge. Thesupernatant is decanted and the resulting DNA pellet is washed twicewith 80% ethanol and dried. This DNA is resuspended in 30 μL of TEbuffer. 20 μL of this sample is loaded on a 1.0% agarose gel andsubjected to electrophoresis in TAE buffer.

In a typical serum assay, 50 μg of DNA in free, encapsulated, orencapsulated+0.5% Triton X100 was aliquoted into 1.5 mL Eppendorf tubes.To the tubes were added 45 μl normal murine or human serum, dH2O (tomake final volume 50 The tubes were sealed with parafilm and incubatedat 37° C. A sample of the free, encapsulated, or encapsulated+0.5%Triton X100 not digested by nuclease (standard) was frozen in liquidnitrogen in an Eppendorf tube and stored at −20° C. Aliquots were takenat various time points, added to GDP buffer containing proteinase K (133μg/mL) and immediately frozen in liquid nitrogen to stop the reaction.Once all of the time points were collected, the samples were incubatedat 55° C. in a waterbath to activate proteinase K enabling it todenature any remaining exonuclease. Proteinase K digested samples wereapplied to polyacrylamide gels to assess levels of exonucleasedegradation.

Particles disclosed above demonstrate serum stability by showing lessthan 5% and preferably undetectable amounts of DNA degradation (partialor total) as a result of such treatment, even in the presence of 100 UDNase 1. This compares favorably to free DNA, which is completelydegraded, and plasmid/lipid complexes (such as DOTMA or DODAC:DOPEcomplexes), wherein DNA is substantially (i.e., greater than 20%, often80%) degraded after such treatment.

Example 5 Characterization of SNALPs

This example describes disease site targeting and gene expressionresulting from intravenous administration of SNALP encapsulatingplasmids in tumor bearing mice.

Plasmid DNA was encapsulated in small (diameter ˜70 nm) nucleicacid-lipid particles (i.e., SNALP) comprising comprise of one plasmidper particle, encapsulated within a lipid bilayer stabilized by thepresence of a bilayer stabilizing component, such as apoly(ethyleneglycol) (PEG) coating. SNALP exhibited extended circulationlifetimes following intravenous administration and promoted delivery ofintact plasmid to distal tumor sites resulting in reporter geneexpression at the disease site.

SNALP with long circulation times accumulated to levels corresponding tofive to ten percent of the total injected dose per gram of tumor orgreater than 1000 copies of plasmid DNA per cell, giving rise to levelsof gene expression that were more than two orders of magnitude greaterthan those observed in any other tissue. Interestingly, although theliver accumulated 20-30% of the total injected dose, very low levels ofgene expression were observed in the liver. This is thought to be due tothe limited hepatocellular uptake of the PEG-ylated SNALP. See, FIGS.8-10.

The in vivo delivery and transfection potential of nucleic acid-lipidparticles containing a bilayer stabilizing component was furtherenhanced through the incorporation of a cationic PEG lipid (CPL)consisting of a DSPE anchor, PEG₃₄₀₀ spacer chain and a cationic headgroup. When CPL were incorporated into SNALP at concentrations of 2 to 4mol % the resulting CPL-SNALP were of a similar size and stability asnative SNALP. Incorporation of CPL resulted in a dramatic increase inintracellular delivery and a concomitant increase in transfectionactivity measured both in vitro and in vivo. Specifically, CPL-SNALPyielded 10⁵-fold more in vitro gene expression than native SNALP. WhenCPL-SNALP were administered intravenously they yielded a substantial(250 fold) increase in hepatic gene expression compared to native SNALP.The increase in CPL-SNALP potency was specific to the liver. The levelsof gene expression measured in the lung, kidney, spleen or heartremained unchanged, contributing to more than two orders of magnitudedifferential in the gene expression measured in the liver vs. otherorgans.

These results illustrate the potential for modulating the deliveryproperties of PEG-lipid containing systems while retaining the stabilityand small uniform size required to achieve systemic gene delivery. Inparticular they demonstrate that disease site targeting and tissuespecific gene expression can be re-programmed by altering the lipidcomposition of non-viral gene delivery systems.

Example 6 SNALPs Containing PEG-DAG Conjugates

This example demonstrates the preparation of a series ofPEG-diacylglycerol lipids (PEG-DAG) SNALPs. In this example, theencapsulated nucleic acid is a plasmid.

PEG-DAG SNALP were prepared incorporating 10 mol percentPEG-dilaurylglycerol (C₁₂), PEG-dimyristylglycerol (C₁₄),PEG-dipalmitoylglycerol (C₁₆) or PEG-disterylglycerol (C₁₈) andevaluated for in vitro transfection activity, pharmacokinetics and thebiodistribution of gene expression resulting from systemicadministration in tumor bearing mice. PEG-DAG lipid containing SNALPdemonstrated a similar relationship between acyl chain length and invitro transfection activity to those containing PEG-ceramides. Shorteracyl chain anchors (dimyristyl (C₁₄) and dipalmitoyl (C₁₆)) resulted inSNALP particles that were less stable but have higher transfectionactivity in vitro than those incorporating longer acyl chain anchors(disteryl (C₁₈)). Evaluation of the pharmacokinetics of PEG-DAGcontaining SNALP confirmed a correlation between the stability of thePEG lipid component and the circulation lifetime of SNALP. SNALPcontaining PEG-dimyristylglycerol (C₁₄), PEG-dipalmitoylglycerol (C₁₆)and PEG-disterylglycerol (C₁₈) demonstrated circulation half-lives of0.75, 7 and 15 hours respectively. Extended circulation lifetime in turncorrelates with an increase in tumor delivery and concomitant geneexpression.

Upon intravenous administration, PEG-disterylglycerol (C₁₈) containingSNALP bypass so-called ‘first pass’ organs, including the lung, andelicited gene expression in distal tumor tissue. The level of reportergene expression observed in tumors represents a 100 to 1000-folddifferential over that observed in any other tissue. This compared wellwith the behavior of SNALP containing PEG-ceramide C₂₀. Theincorporation of PEG-DAG in SNALP confirmed that small size, low surfacecharge and extended circulation lifetimes are prerequisite to thepassive disease site targeting leading to accumulation of plasmid DNAand gene expression in tumors following systemic administration ofnon-viral transfection systems. See, FIGS. 5-10.

Materials and Methods

Materials

DOPE and DSPC were obtained from Northern Lipids (Vancouver, BC). DODACand the PEG-diacylglycerols were manufactured by Inex Pharmaceuticals(Burnaby, BC). The other materials, HEPES, OGP and ³H-cholesterylhexadecyl ether, were obtained from a number of different commercialsources.

DOPE:DODAC:PEG-Diacylglycerols (82.5:7.5:10) large unilamellar vesicleswere prepared via detergent dialysis in Hepes Buffered Saline (150 mMNaCl and 10 mM HEPES) for 48 hours. Lipid stock solutions were preparedin ethanol and then dried down to create a lipid film which wasreconstituted in final 200 mM OGP. LUVs were labeled with ³H-cholesterylhexadecyl ether at 1 uCi/1 mg lipid. Particle sizes were determined bynicomp analysis. Radioactivity was determined by scintillation countingwith Picofluor20.

SNALP containing PEG-Diacyglycerols were formulated via detergentdialysis by varying the salt concentration to maximize the percent ofDNA encapsulation. Optimal salt concentration was chosen for the 48 hourdetergent dialysis. Empty vesicles were removed by one step sucrosecentrifugation. 3.5% sucrose was used to separate out the emptyparticles from the plasmid-containing PEG-Diacylglycerol formulationsexcept for PEG-Dimyristylglycerol containing SNALP which used 5.0%sucrose. Empty vesicles migrated to the top of the tube which werefractioned out and removed.

In Vitro Transfection

5×10⁴ cells/ml were plated onto 24-well plates (1 ml). Cells were leftto grow for 24 hours. 500 μl of transfection media (2.5 μg/well) wasadded and then incubated for stated timepoints. Transfection media wasaspirated after timepoint and then exposed to complete media for another24 hours at 37° C. in 5.0% CO₂. Complete media was removed. Cells werewashed with PBS twice and stored at −70° C. until day of experiment.Cells were lysed with 150 μl of 1×CCLR containing protease inhibitors.Plates were shaken for 5 minutes. 20 μl of each sample were assayed induplicate on a 96-well luminescence plate for luciferase activity.

Pharmacokinetics, Biodistribution, and In Vivo Gene Expression

Pharmacokinetics and biodistribution were all determined by normalizingthe data to the quantity of radioactivity present. Approximately 500 μlof blood was obtained by cardiac puncture. Red blood cells and plasmawere separated by centrifugation (4° C., 3000 rpm, 10 minutes) and 100μl of plasma was used to determine radioactive counts. Organs wereharvested at specified timepoints and homogenized in lysing matrix tubes(Fast Prep, 2×15 seconds, 4.5 intensity) to assay a portion of themixture.

Gene expression was determined by luciferase assay. Organs wereharvested, homogenized, and kept on ice throughout the experiment.Lysates were centrifuged (10,000 rpm, 5 minutes) and 20 μl ofsupernatant were assayed in duplicate on a 96-well luminescence platefor luciferase activity. The results are depicted in FIGS. 7-10.

In Vitro Gene Silencing

Cells were transfected with SPLP comprising PEG-lipid conjugates andcontaining a plasmid encoding luciferase under the control of the CMVpromoter and SNALPs containing anti-luciferase siRNA, according to themethods described above. Gene expression was determined by luciferaseassay. The results are depicted in FIG. 17.

Example 7 Expression of Nucleic Acids Encapsulated in SPLP ComprisingPEG-Dialkyloxypropyl Conjugates

This example describes experiments comparing expression of nucleic acidsencapsulated in SPLP comprising PEG-dialkyloxypropyl conjugates. AllSPLP formulations comprise a plasmid encoding luiferase under thecontrol of the CMV promoter (pLO55).

# # Group Mice Tumor Route Treatment Route Doses Timepoint ASSAY*** A 4Neuro- SC PBS IV 1 48 hrs Body weights, 2a Blood analyses, B 5 Neuro- SCSPLP PEG-DSG IV 1 48 hrs Luciferase 2a activity C 5 Neuro- SC SPLPPEG-A-DSA IV 1 48 hrs 2a D 5 Neuro- SC SPLP PEG-A-DPA IV 1 48 hrs 2a E 5Neuro- SC SPLP PEG-A-DMA IV 1 48 hrs 2a

The lipids (DSPC:CHOL:DODMA:PEG-Lipid) were present in the SPLP in thefollowing molar ratios (20:55:15:10). The following formulations weremade:

A: PBS sterile filtered, 5 mL.

B: pL055-SPLP with PEG-DSG, 2 mL at 0.50 mg/mL.

C: pL055-SPLP with PEG-A-DSA, 2 mL at 0.50 mg/mL.

D: pL055-SPLP with PEG-A-DPA, 2 mL at 0.50 mg/mL.

E: pL055-SPLP with PEG-A-DMA, 2 mL at 0.50 mg/mL.

# Seeding Injection Collection Group Mice date Treatment date date A 4Day 0 PBS Day 12 Day 14 B 5 Day 0 SPLP PEG-DSG Day 12 Day 14 C 5 Day 0SPLP PEG-A-DSA Day 12 Day 14 D 5 Day 0 SPLP PEG-A-DPA Day 12 Day 14 E 5Day 0 SPLP PEG-A-DMA Day 12 Day 14

1.5×10⁶Neuro2A cells were administered to each mouse on day 0. When thetumors were of a suitable size (200-400 mm³), mice were randomized andtreated with one dose of an SPLP formulation or PBS by intravenous (IV)injection. Dose amounts are based on body weight measurements taken onthe day of dosing. 48 hours after SPLP administration, the mice weresacrificed, their blood was collected, and the following tissues werecollected weighed, immediately frozen and stored at −80° C. untilfurther analysis: tumor, liver (cut in 2 halves), lungs, spleen andheart.

Gene expression in collected tissues was determined by assaying forenzymatic activity of expressed luciferase reporter protein. The resultsare shown in FIGS. 11 and 12.

The results indicate that SPLP comprising PEG-dialkyloxypropyls (i.e.,PEG-DAA) can conveniently be used to transfect distal tumor tosubstantially the same extent as SPLP comprising PEG-diacylglycerols.Moreover, the transfection levels seen with SPLP containingPEG-dialkyloxypropyl are similar to those seen with SPLP containingPEG-diacylglycerols (e.g. PEG-DSG). It was also shown that similar tothe PEG-diacylglycerol system, very little transfection occurred innon-tumor tissues. Moreover, the SPLP comprising PEG-dialkyloxypropylsexhibit reduced toxicity compared to other SPLP formulations.

Example 8 SNALPs Containing PEG-Dialkyloxypropyl Conjugates

This example described experiments analyzing the biodistribution (localand systemic) and pharmacokinetics of a series of PEG-dialkyloxypropyllipids SNALPs (i.e., SPLP containing encapsulated siRNA.

Local Biodistribution

To determine the local distribution of SPLP resulting from systemicadministration of anti-β galactosidase siRNA containing SNALP inNeuro-2a tumor bearing mice via fluorescent microscopy.

A: PBS

B: anti-βgal siRNA-Rhodamine-PE labeled-DSPC:Chol:DODMA:PEG-A-DMA SNALP(1:20:54:15:10)

Group Mice Cells Treatment Timepoint Assay A 2 Neuro2A PBS 24 hrFluorescent Photomicro- scopy B 5 Neuro2A anti-Bgal siRNA- 24 hrFluorescent Rhodamine-PE labeled- DSPC:Chol:DODMA: Photomicro- PEG-A-DMAscopy

1.5×10⁶ Neuro2A cells were administered to each mouse on day 0. When thetumors were of a suitable size (200-400 mm³, typically day 9-12)), micewere randomized and treated with one dose of an SNALP formulationcomprising 100 μg siRNA or PBS by intravenous (IV) injection in a totalvolume of 230 μl. Dose amounts are based on body weight measurementstaken on the day of dosing. 24 hours after SPLP administration, the micewere sacrificed, their blood was collected, and the following tissueswere collected weighed, immediately frozen and stored at −80 C. untilfurther analysis: tumor, liver (cut in 2 halves), lungs, spleen & heart.

Local distribution of the SNALP was determined by fluorescencemicroscopy. Accumulation of SNALP is seen in, e.g., the liver,demonstrating the SNALP comprising PEG-dialkyloxypropyls are able toextravasate, i.e., exit the circulation and home to a target tissue ororgan.

Pharmacokinetics and Systemic Biodistribution

This example illustrates the pharmacokinetics and biodistribution ofSPLPs containing a plasmid encoding luciferase under the control of theCMV promoter (LOSS) and SNALPs containing anti-luciferase siRNA in miceseeded subcutaneously with Neuro2A tumors.

Group Mice Cells Treatment Timepoint (h) A 6 Neuro2A[3-H]CHE-L055-DSPC:Chol: 0.25, 1, 4, 8, 24 DODMA:PEG-A-DMA B 6 Neuro2A[3-H]CHE-anti-luc siRNA- 0.25, 1, 4, 8, 24 DSPC:Chol:DODMA: PEG-A-DMA C6 Neuro2A [3-H]CHE-L055 -DSPC:Chol: 0.25, 1, 4, 8, 24 DODMA:PEG-C-DMA D6 Neuro2A [3-H]CHE-L055-pSPLP (PEI) 0.25, 1, 4, 8, 24 E 6 Neuro2A[3-H]CHE-L055-DSPC:Chol: 0.25, 1, 4, 8, 24 DODMA:PEG-DSG

All samples are to be provided at 0.5 mg/ml nucleic acid. The followingSPLP and SNALP formulations were prepared:

A. [³H] CHE-L055-DSPC:Chol:DODMA:PEG-A-DMA (20:55:15:10)

B. [³H] CHE-anti-luc siRNA-DSPC:Chol:DODMA:PEG-A-DMA (20:55:15:10)

C. [³H] CHE-L055-DSPC:Chol:DODMA:PEG-C-DMA (20:55:15:10)

D. [³H] CHE-L055-pSPLP (PEI) (i.e., precondensed SPLP)

E. [³H] CHE-L055-DSPC:Chol:DODMA:PEG-DSG (20:55:15:10)

# Seeding Injection Collection Group Mice date Treatment date date A 6Day 0 [3-H]CHE-L055-DSPC: Day 12 July 31 Chol:DODMA:PEG-A-DMA B 6 Day 0[3-H]CHE-anti-luc siRNA- Day 12 July 31 DSPC:Chol:DODMA: PEG-A-DMA C 6Day 0 [3-H]CHE-L055 -DSPC: Day 13 Day 14 Chol:DODMA:PEG-C-DMA D 6 Day 0[3-H]CHE-L055-pSPLP Day 13 Day 14 (PEI) E 6 Day 0 [3-H]CHE-L055-DSPC:Day 14 Day 15 Chol:DODMA:PEG-DSG

30 male A/J mice (Jackson Laboratories) were seeded subcutaneously withNeuro 2A cells at a dose of 1.5×10⁶ cells in a total volume of 50 μLphosphate buffered saline on day zero. After tumors reached appropriatesize (typically on day 9 or later), 200 μl (100 μm nucleic acid) of theSPLP or SNALP preparations described above, were administeredintravenously. 0.25, 1, 2, 4, and 8 hours after administration of SPLPor SNALP, mice were weighed and blood (typically 25 μL) was collected bytail nick. 24 hours after administration of SPLP or SNALP, mice weresacrificed, blood was collected and assayed for clearance of [³H]CHE.Organs (e.g., liver, lung, spleen, kidney, heart) and tumors werecollected and evaluated for [³H]CHE accumulation. The results are shownin FIGS. 13-16.

For all formulations, SPLP containing PEG-DSG remained in circulationthe longest, with 50% of the injected dose remaining after 6 h.Interestingly, there appeared to be a initial rapid clearance of pSPLPwithin the first 15 minutes that was not seen for any other formulation.After 1 h the clearance profile of the pSPLP was quite similar to SPLP.This initial rapid clearance for the pSPLP sample may indicate thatthere are actually two types of particles present, one that clears veryrapidly and one that behaves very much like SPLP.

Anti-Luc siRNA containing vesicles (SNALP) formulated with the C14PEG-A-DMA showed more rapid clearance from blood than SPLP containingthe C18 PEG-DSG. However, this SNALP formulation showed significantlyslower blood clearance than SPLP formulated with the same PEG lipid. Apossible reason for this result maybe that siRNA containing particlescan evade the cellular immune system more readily than plasmidcontaining SPLP.

SPLP comprising PEG-C-DMA demonstrated a rapid clearance from blood,which was substantially the same as that observed for SPLP comprisingPEG-A-DMA. For both of these formulations, the plasma half lives wereapproximately 2 h, lower than for SPLP containing C18 PEG-lipids.

SPLP containing PEG-DSG had the highest tumor accumulation at 10.9%inject dose per gram tissue. The two SPLP formulations containing theC14 PEG-lipids, PEG-A-DMA and PEG-C-DMA, had much lower tumoraccumulation of 6.1% and 5.9% injected dose per gram tissue. The SiRNASNALP had slightly more tumor accumulation than an SPLP sample with thesame PEG-lipid at 7.3%, which also correlates relatively well with theplasma half-life for this SNALP. The pSPLP formulation had tumoraccumulation at 7.5%, which is lower than the comparable PEG-DSG SPLP.

Accumulation of PEG-DSG containing SPLP and pSPLP in the heart and lungswas higher than the other SPLP and SNALP, which is consistent with theincreased circulation half lives of particles with C18 PEG-lipids. Notsurprisingly, there was an inverse relationship between plasma half-lifeand accumulation in the liver for all samples tested, while no trend wasapparent for sample accumulation in the spleen. Accumulation in thekidneys was very low for all formulations tested, with accumulationbetween 1.2 and 2.4% injected dose per gram tissue.

Example 9 Silencing of Gene Expression with SNALPS

This example illustrates silencing of gene expression in Neuro 2A tumorbearing mice after co-administration of SPLPs containing a plasmidencoding luciferase under the control of the CMV promoter and SNALPscontaining anti-luciferase siRNA.

# # Group Mice Tumor Route Treatment Timepoint Route Doses 1 3 Neuro- SQPBS/PBS 48 h IV 1 24A 4 2a L055-SPLP/PBS mix 24 h 24B 4L055-SPLP/anti-luc siRNA liposomes mix 48A 4 L055-SPLP/PBS mix 48 h 48B4 L055-SPLP/anti-luc siRNA liposomes mix 72A 4 L055-SPLP/PBS mix 72 h72B 4 L055-SPLP/anti-luc siRNA liposomes mix

# Seeding Injection Collection Group Mice Date Route IV TreatmentTimepoint date Date 1 3 Day 0 SQ PBS/PBS 48 h Day 13 Day 15 24A 4L055-SPLP/PBS mix 24 h Day 14 24B 4 L055-SPLP/anti-luc Day 14 siRNAliposomes mix 48A 4 L055-SPLP/PBS mix 48 h Day 13 48B 4L055-SPLP/anti-luc Day 13 siRNA liposomes mix 72A 4 L055-SPLP/PBS mix 72h Day 12 72B 4 L055-SPLP/anti-luc Day 12 siRNA liposomes mix

36 male A/J mice (Jackson Laboratories) were seeded subcutaneously withNeuro 2A cells at a dose of 1.5×10⁶ cells in a total volume of 50 μLphosphate buffered saline on day zero. Once tumors reached appropriatesize (typically on day 9 or later), 200-240 μl PBS, SPLP, or SNALPformulations (100 μg nucleic acid total) prepared as described inExample 6 above, were administered intravenously. 24, 48, or 72 afteradministration of PBS, SPLP or a mixture of SPLP and SNALP, mice weresacrificed and organs (e.g., liver, lung, spleen, kidney, heart) andtumors were collected and evaluated for luciferase activity. The resultsare shown in FIGS. 18-22.

The results demonstrate that co-administration of pL055 SPLP andanti-luc siRNA SNALP (both containing PEG-A-DMA) maximally decreasesluciferase gene expression by 40% forty-eight hours after a single ivdose.

Example 10 Down Regulation of β-Gal Activity in Stably TransfectedCT26-CL25 Cells

SNALP were prepared containing siRNA duplex directed against theβ-Galactosidase reporter gene and applied to the β-galactosidaseexpressing stable cell line: CT26CL25, plated at 2×10⁴ cells/well at aconcentration of 1.0 μg/mL siRNA. Cells were exposed to SNALP for 24hours and β-galactosidase activity was determined after 96 hours.Silencing was observed in 90% of the cells in culture which correlateswith silencing of a target protein in 40% of cells in vivo.

Example 11 Liver Distribution of Rhodamine Labeled SNALP Following aSingle Intravenous Administration

SNALP were prepared containing siRNA duplex directed against theβ-Galactosidase reporter gene using and administered to A/J miceintravenously, through the tail vein. Tissues were collected at 24hours, snap frozen and sectioned for visualization of SNALPdissemination. Cells were stained with rhodamine and counterstained withDAPI, which stains nuclei. The in vivo biodistribution of the SNALPfavors the liver, with as much as 50% of the administered SNALP materialdelivered to the liver. The SNALP delivered to the liver is found in adiffuse pattern, distributed throughout the liver.

Example 12 Silencing of Gene Expression Following Delivery of siRNAEncapsulated in SPLP Comprising Cationic Lipids

This example describes experiments comparing expression of nucleic acidsfollowing in vitro transfection of Neuro2A cells with SNALP comprising:(1) DODAC, DODMA, or DLinDMA; (2) PEG-C-DMA; and (3) an siRNA duplexdirected against luciferase encapsulated in the SNALP (i.e., siRNAcomprising the following sequence: GAUUAUGUCCGGUUAUGUAUU (SEQ ID NO:1)and targeting the DNA sequence complementary to: GATTATGTCCGGTTATGTATT(SEQ ID NO:3)). Neuro2A cells were stably transfected with a plasmidencoding luciferase under the control of the CMV promoter (pLO55). Thestably transfected cells were then transfected with SNALP comprising:15, 20, 25, 30, 35, or 40% of DODAC, DODMA, or DLinDMA; 2% PEG-C-DMA,and an siRNA duplex directed against luciferase encapsulated in theSNALP. Luciferase protein expression was measured 48 hours aftertransfection with SNALP. SNALP comprising 30% DLinDMA was more effectivein reducing luciferase expression in the Neuro2A cells than SNALPcomprising DODAC or DODMA were. These results are shown in FIG. 23.

DLinDMA, the most fusogenic lipid with the lowest apparent phasetransition temperature, yielded the greatest knockdown when incorporatedin SNALP, with luciferase expression only 21% that of the untreatedcontrol. This was followed by the DLenDMA formulation (32%), and DODMA(54%). The close correspondence between knockdown efficiency and theH_(II) phase forming ability of the cationic lipid as observed suggeststhat the two parameters are linked.

Example 13 SNALP Containing Unsaturated Cationic Lipids Show IncreasedGene-Silencing Activity

The ability of SNALP containing each of the four cationic lipids (i.e.,DSDMA, DODMA, DLinDMA, and DLenDMA) to effect gene silencing in stablytransfected Neuro2A cells was evaluated. Neuro2A cells stablytransfected to express the luciferase were treated with SNALP containinganti-luciferase siRNA for 48 hours. Gene-silencing efficiency wasevaluated by comparing the remaining luciferase activity in these cellsto that remaining in cells treated with control SNALP containingmismatch siRNA.

Formulations comprising the saturated lipid DSDMA demonstrated noactivity. As unsaturation in the lipid's alkyl chain increased, so didthe capacity for RNA interference, with DLinDMA particles yielding an80% knockdown in gene expression. ³¹P-NMR established DLinDMA as havingthe lowest phase transition temperature in the series and accordingly,being the most fusogenic lipid. Particles comprising DLenDMA, the mostunsaturated lipid, were slightly less efficient than those containingDLinDMA. All results were found to be significant by t-Test (P<0.05 atsiRNA concentration of 0.5 μg/mL, and P<0.01 at siRNA concentration of1.0 μg/mL). Error bars represent standard deviation, n=3. The resultsare shown in FIG. 24.

Example 14 In Vivo Transfection of Organs by Various SPLP Formulations

This example describes experiments demonstrating in vivo transfection oforgans with that SPLP comprising 15% DLinDMA can be used SPLPencapsulating a plasmid encoding luciferase under the control of the CMVpromoter were administered to Neuro2A tumor bearing male A/J mice. TheSPLP had the following formulations:

Sample Description A SPLP-PEG₂₀₀₀-C-DMA (CHOL:DSPC:DODMA:PEG₂₀₀₀-C-DMA55:20:15:10 mol %) B SPLP-PEG₂₀₀₀ DlinDMA(CHOL:DSPC:DlinDMA:PEG₂₀₀₀-C-DMA 55:20:15:10 mol %) CSPLP-PEG₇₅₀-C-DMA/DODMA (CHOL:DSPC:DODMA:PEG₇₅₀-C-DMA 55:20:15:10 mol %)D SPLP-PEG₇₅₀-C-DMA/DLinDMA (CHOL:DSPC:DlinDMA:PEG₇₅₀-C-DMA 55:20:15:10mol %) 0.41 mg/ml E SPLP- High PEG₇₅₀-C-DMA(CHOL:DSPC:DODMA:PEG₇₅₀-C-DMA 50:20:15:15 mol %) F SPLP- HighPEG₇₅₀-C-DMA (CHOL:DSPC:DlinDMA:PEG₇₅₀-C-DMA 50:20:15:15 mol %) GSPLP-DODAC (CHOL:DSPC:DODMA:PEG₂₀₀₀-C-DMA:DODAC 45:20:15:10:10 mol %)0.35 mg/ml

Luciferase gene expression was assessed in liver, lung, spleen, heartand tumors 48 hours after intravenous administration of the SPLP. Theresults are shown in FIG. 25.

Example 15 In Vivo Transfection of Tumor by Additional SPLP Formulations

This example describes experiments demonstrating in vivo transfection oforgans with that SPLP comprising DLinDMA or DODMA and varyingpercentages (15%, 10%, 5%, or 2.5%) of PEG-C-DMA. SPLP encapsulating aplasmid encoding luciferase were administered to Neuro2A tumor bearingmale A/J mice. The SPLP had the following formulations:

Mol % (DSPC:Chol:PEG-C-DMA:DXDMA A 20:50:15:15 (DODMA) B 20:55:10:15(DODMA) C 20:60:5:15 (DODMA) D 20:62.5:2.5:15 (DODMA) E 20:55:10:15(DLinDMA) F 20:60:5:15 (DLinDMA) G 20:62.5:2.5:15 (DLinDMA)

Luciferase gene expression was assessed in tumors 48 hours afterintravenous administration of SPLP. The results are shown in FIG. 26.

Example 16 Blood Clearance of Lipid Vesicles Comprising PEG-C-DMA

This example describes experiments conducted to assess the bloodclearance rate of lipid vesicles comprising various percentages ofPEG-C-DMA. A single intravenous dose of ³H—CHE-labeled SPLP, SNALP, orempty vesicles was administered to male A/J mice. SPLP comprised thecationic lipid DODMA and SNALP comprised the cationic lipid DLinDMA. Thelipid vesicles had the following formulations:

Mol % (DSPC:Chol:PEG- Group Treatment C-DMA:Cationic Lipid) A Emptyvesicles 20:48:2:30 B SNALP (DlinDMA, PEG-C-DMA) 20:48:2:30 C SNALP(DlinDMA, PEG-C-DMA) 20:55:5:20 D SPLP (15 mol % PEG-C-DMA) 20:50:15:15E SPLP (10 mol % PEG-C-DMA) 20:55:10:15 F SPLP (5 mol % PEG-C-DMA)20:60:5:15

The percentage of the injected dose of lipid vesicle remaining in plasmaof the mice was determined at 1, 2, 4, and 24 hours following theadministration of the ³H—CHE-labeled SPLP, SNALP, or empty vesicles. Theresults are shown in FIG. 27.

Example 17 Biodistribution of Lipid Vesicles Comprising PEG-C-DMA

The example describes experiments conducted to assess thebiodistribution of lipid vesicles comprising various percentages ofPEG-C-DMA. A single intravenous dose of ³H—CHE-labeled SPLP, SNALP, orempty vesicles was administered to Neuro 2A tumor bearing male A/J mice.SPLP comprised the cationic lipid DODMA and SNALP comprised the cationiclipid DLinDMA. The lipid vesicles had the following formulations:

Mol % (DSPC:Chol:PEG- Group Treatment C-DMA:Cationic Lipid) A Emptyvesicles 20:48:2:30 B SNALP (DlinDMA, PEG-C-DMA) 20:48:2:30 C SNALP(DlinDMA, PEG-C-DMA) 20:55:5:20 D SPLP (15 mol % PEG-C-DMA) 20:50:15:15E SPLP (10 mol % PEG-C-DMA) 20:55:10:15 F SPLP (5 mol % PEG-C-DMA)20:60:5:15

The percentage of the injected dose of lipid vesicles was assessed inthe liver, spleen, lungs, and tumor of the mice 48 hours afteradministration of the ³H—CHE-labeled vesicles. The results are shown inFIG. 28.

Example 18 Silencing of Gene Expression at a Distal Tumor

This example describes experiments demonstrating gene silencing indistal tumors following administration of SNALP comprising DLinDMA andencapsulating an anti-luciferase siRNA sequence.

Neuro 2A cells were stably transfected with a plasmid encodingluciferase under the control of the CMV promoter (pLO55) to generateNeuro 2A-G cells. Male A/J mice were seeded with the Neuro 2A-G cells.The SNALP encapsulating the anti-luciferase siRNA sequence (i.e., siRNAcomprising the following sequence: GAUUAUGUCCGGUUAUGUAUU (SEQ ID NO:1)and targeting the DNA sequence complementary to: GATTATGTCCGGTTATGTATT(SEQ ID NO:3)) were administered to the Neuro2A-G tumor bearing A/J miceintravenously. The SNALP formulations were as follows:

Mol % (DSPC:Chol:PEG- Group C-DAA:DLinDMA) PBS A Anti Luciferase SNALP20:48:2:30 B Control (Invert Sequence) SNALP 20:48:2:30 C AntiLuciferase SNALP 20:55:5:20 D Control (Invert Sequence) SNALP 20:55:5:20E Anti Luciferase SNALP 20:55:10:15 F Control (Invert Sequence) SNALP20:55:10:15

Luciferase gene expression was measured 48 hours followingadministration of SNALP comprising DLinDMA and encapsulating ananti-luciferase siRNA sequence. The results are shown in FIG. 29.

Example 19 Silencing of Gene Expression in Neuro2A-G Tumor Cells InVitro

This example describes experiments demonstrating gene silencing inmammalian cells following contact with SNALP comprising DLinDMA andencapsulating an anti-luciferase siRNA sequence described in Example 3above. Neuro 2A cells were stably transfected with a plasmid encodingluciferase as described in Example 3 above to generate Neuro 2A-G cells.The Neuro 2A-G cell were contacted with SNALP formulations for 24 or 48hours. The SNALP formulations comprised either PEG-C-DLA (C₁₂) orPEG-C-DMA (C₁₄) and are as follows:

Mol % (DSPC:Chol:PEG- Group Treatment C-DAA:DLinDMA) A SNALP (PEG-C-DLA)20:48:2:30 B SNALP (PEG-C-DLA) 20:45:5:30 C SNALP (PEG-C-DLA)20:40:10:30 D SNALP (PEG-C-DMA) 20:48:2:30

Luciferase gene expression was measured 24 or 48 hours followingcontacting the Neuro 2A-G cells with SNALP encapsulating ananti-luciferase siRNA sequence. The results are shown in FIG. 30.

Example 20 Silencing of Gene Expression in Neuro2A-G Tumor Cells InVitro

This example describes experiments demonstrating gene silencing inmammalian cells following contact with SNALP comprising DLinDMA andencapsulating an anti-luciferase siRNA sequence described in Example 3above. Neuro 2A cells were stably transfected with a plasmid encodingluciferase as described in Example 3 above to generate Neuro 2A-G cells.The Neuro 2A-G cells were contacted with SNALP formulations for 48 hoursin the presence and absence of chloroquine. The SNALP formulationscontained varying percentages of PEG-C-DMA (C₁₄) and either DODMA orDLinDMA. The formulation were as follows:

Mol % (DSPC:Chol:PEG- Group Treatment C-DAA:DLinDMA) A PBS — B NakedsiRNA — C SNALP (PEG-C-DMA) 20:40:10:30 D SNALP (PEG-C-DMA) 20:46:4:30 ESNALP (PEG-C-DMA) 20:48:2:30 F SNALP (PEG-C-DMA) 20:49:1:30

Luciferase gene expression was measured 48 hours following contactingthe Neuro 2A-G cells with the SNALP encapsulating an anti-luciferasesiRNA sequence. The results are shown in FIG. 31.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents and PCT publications,and Genbank Accession Nos. are herein incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of introducing an interfering RNA into acell, said method comprising contacting said cell with a nucleicacid-lipid particle comprising (a) said interfering RNA; (b) a cationiclipid of Formula I having the following structure:

wherein: R¹ and R² are independently selected from the group consistingof: H and C₁-C₃ alkyl; and R³ and R⁴ are independently selected from thegroup consisting of alkyl groups having from about 10 to about 20 carbonatoms, wherein at least one of R³ and R⁴ comprises at least two sites ofunsaturation; (c) a non-cationic lipid; and (d) a conjugated lipid thatinhibits aggregation of particles.
 2. The method of claim 1, whereinsaid interfering RNA in said nucleic acid-lipid particle is resistant inaqueous solution to degradation with a nuclease.
 3. The method of claim1, wherein said particle has a median diameter of less than about 150nm.
 4. The method of claim 1, wherein said interfering RNA comprises asmall interfering RNA (siRNA).
 5. The method of claim 1, wherein saidinterfering RNA is transcribed from a plasmid.
 6. The method of claim 1,wherein said non-cationic lipid is a member selected from the groupconsisting of dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine(EPC), di stearoylphosphatidylcholine (DSPC),palmitoyloleyolphosphatidylglycerol (POPG), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,palmitoyloleoyl-phosphatidylethanolamine (POPE),1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, and amixture thereof.
 7. The method of claim 1, wherein the conjugated lipidthat inhibits aggregation of particles is a member selected from thegroup consisting of a polyethyleneglycol (PEG)-lipid conjugate, apolyamide (ATTA)-lipid conjugate, and a mixture thereof.
 8. The methodof claim 1, wherein the conjugated lipid that inhibits aggregation ofparticles is a polyethyleneglycol (PEG)-lipid.
 9. The method of claim 8,wherein the PEG-lipid is member selected from the group consisting of aPEG-diacylglycerol, a PEG-dialkyloxypropyl, a PEG-phospholipid, aPEG-ceramide, and a mixture thereof.
 10. The method of claim 1, whereinthe conjugated lipid that inhibits aggregation of particles is apolyethyleneglycol (PEG)-dialkyloxypropyl conjugate.
 11. The method ofclaim 10, wherein the PEG-dialkyloxypropyl conjugate isPEG-dimyristyloxypropyl (C₁₄).
 12. The method of claim 1, wherein saidcell is in a mammal.
 13. The method of claim 12, wherein presence ofsaid interfering RNA in said cell is detectable for at least 48 hoursafter said contacting.
 14. The method of claim 12, wherein presence ofsaid interfering RNA in said cell is detectable for at least 24 hoursafter said contacting.